Treatment method

ABSTRACT

The present invention relates to methods of treating a disease, and methods for reduction of the formation of anti-drug antibodies (ADAs) in response to the administration of a therapeutic agent. The invention further relates to methods of treating a disease, particularly a B-cell proliferative disorder, and methods for reduction of adverse effects in response to the administration of a therapeutic agent, particularly a T-cell activating therapeutic agent.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.17/748,707, filed May 19, 2022, which is a divisional of U.S. patentapplication Ser. No. 17/519,790, filed Nov. 5, 2021, which is adivisional of U.S. patent application Ser. No. 17/240,821, filed Apr.26, 2021, which is a divisional of U.S. patent application Ser. No.15/371,891, filed Dec. 7, 2016, now U.S. Pat. No. 11,013,801, whichclaims priority to European Patent Application No. EP 16193151.4, filedOct. 10, 2016, European Patent Application No. EP 16172739.1, filed Jun.2, 2016, and European Patent Application No. EP 15198715.3, filed Dec.9, 2015, the disclosures of which are incorporated herein by referencein their entirety.

SEQUENCE LISTING

The present application contains a Sequence Listing which has beensubmitted in XML format and is hereby incorporated by reference in itsentirety. Said XML copy, created on Nov. 10, 2022, is named51177-018005_Sequence_Listing_11_10_22 and is 169,634 bytes in size.

FIELD OF THE INVENTION

The present invention relates to methods of treating a disease, andmethods for reduction of the formation of anti-drug antibodies (ADAs) inresponse to the administration of a therapeutic agent. The inventionfurther relates to methods of treating a disease, particularly a B-cellproliferative disorder, and methods for reduction of adverse effects inresponse to the administration of a therapeutic agent, particularly aT-cell activating therapeutic agent.

BACKGROUND

The number of biotechnology-derived therapeutic agents available for usein clinical settings has dramatically increased in recent years, andincludes recombinant human cytokines (e.g. α and β interferon,interleukin-2), cellular growth factors (e.g. GM-CSF), hormones (e.g.glucagon), neuromuscular antagonists (e.g. botulinum toxin), bloodproducts (e.g. clotting factor VIII), recombinant receptors (e.g.etanercept) and monoclonal antibodies. Although therapeutic proteins aregenerally considered safe and non-toxic, antibodies against thesetherapeutic agents, known as anti-drug antibodies (ADAs), can developduring treatment. ADAs have been observed in connection with varioustherapeutic agents, such as erythropoietin, factor VIII, insulin,immunotoxins and monoclonal antibodies (Schellekens and Casadevall, JNeurol (2004), 251 [Suppl 2]:II/4-II/9; Mossoba et al., Clin Cancer Res(2011) 17(11): 3697-3705; Hsu et al., British Journal of Dermatology(2014) 170, 261-273). ADA formation is frequent for example inautoimmune patients treated with TNF blockers and impacts clinicaloutcome (Schaeverbecke et al., Rheumatology (2015) doi:10.1093/rheumatology/kev277).

The development of ADAs may influence serum concentrations and functionof therapeutic agents. The presence of ADAs may increase clearance ofthe therapeutic agent through formation of immune complexes betweentherapeutic agent and antibody (neutralizing, non-neutralizing or both),thus reducing the therapeutic agent's half-life. Furthermore, theactivity and effectiveness of the therapeutic agent may be decreasedthrough binding of antibody to the therapeutic agent. ADAs can also beassociated with allergic or hypersensitivity reactions and other adverseevents.

Since these adverse events associated with immune responses caninfluence the safety and efficacy profile of therapeutics,identification and development of strategies to overcome or inhibit ADAsis of great interest.

Several protein engineering approaches have been investigated to reducethe immunogenicity of protein therapeutics, including for examplemasking or alteration of protein B cell epitopes or modification ofprotein T cell epitopes. However, clinical safety and success of theseapproaches has not been tested and will require a significant degree oftime to evaluate. Therefore, there exists an immediate need to developnew interventions using FDA-approved reagents to prevent ADA responses.

Chemotherapy-based approaches aimed at host immune suppression have beenreported (Mossoba et al., Clin Cancer Res (2011) 17(11): 3697-3705).

The anti-CD20 antibody rituximab has been used in combination withmethotrexate and intravenous immune globulin to achieve tolerance toenzyme replacement therapy in a Morbus Pompe patient (Mendelsohn et al.,NEJM (2009) 360:2, 194-195). However, in a clinical trial, hostpretreatment with rituximab did not inhibit the human immune responseagainst the immunotoxin LMB-1 (Hassan et al., Clin Cancer Res (2004) 10,16-18).

B-cell proliferative disorders describe a heterogeneous group ofmalignancies that includes both leukemias and lymphomas. Lymphomasdevelop from lymphatic cells and include two main categories: Hodgkinlymphomas (HL) and the non-Hodgkin lymphomas (NHL). In the UnitedStates, lymphomas of B cell origin constitute approximately 80-85% ofall non-Hodgkin lymphoma cases, and there is considerable heterogeneitywithin the B-cell subset, based upon genotypic and phenotypic expressionpatterns in the B-cell of origin. For example, B cell lymphoma subsetsinclude the slow-growing indolent and incurable diseases, such asFollicular lymphoma (FL) or chronic lymphocytic leukemia (CLL), as wellas the more aggressive subtypes, mantle cell lymphoma (MCL) and diffuselarge B cell lymphoma (DLBCL).

Despite the availability of various agents for the treatment of B-cellproliferative disorders, there is an ongoing need for development ofsafe and effective therapies to prolong remission and improve cure ratesin patients.

A strategy currently being investigated is the engagement of T cellsagainst malignant B cells. In order to effectively engage T cellsagainst malignant B cells, two recent approaches have been developed.These two approaches are: 1) the administration of T cells engineered exvivo to recognize tumour cells (also known as chimeric antigenreceptor-modified T cell therapy [CAR-T cells]) (Maude et al., N Engl JMed (2014) 371, 1507-1517); and, 2) the administration of agents thatactivate endogenous T cells, such as bispecific antibodies (Oak andBartlett, Expert Opin Investig Drugs (2015) 24, 715-724).

An example of the first approach is reported in the study by Maude etal., in which 30 adult and pediatric patients were treated withautologous T cells transduced with a CD19-directed chimeric antigenreceptor lentiviral vector (CTL019 CAR-T cells). The result was asustained remission based upon a 6-month event-free survival rate of 67%and an overall survival rate of 78%. However, all patients had cytokinerelease syndrome (CRS) (associated with tumour burden), with 27% ofpatients having severe CRS. Central nervous system toxicities of unknowncause were also noted at high frequencies.

In contrast, the second approach, which involves activating endogenous Tcells to recognize tumour targets, bypasses this hurdle of scalability,and can also provide competitive efficacy, safety data and potentiallylong term durations of response. In different CD20⁺ hematologicmalignancies, this approach is best exemplified by blinatumomab, a CD19CD3 targeting T cell bispecific molecule (Bargou et al., Science (2008)321, 974-977) that was recently approved for patients with minimalresidual disease-positive acute lymphocytic leukemia (ALL). Thiscompound, which is composed of two single chain Fv fragments (the socalled BiTE® format), directs the lysis of CD19⁺ cells by cytolytic Tcells. The primary constraint of blinatumomab is its short half-life(approximately 2 hours), which necessitates continuous infusion via apump over 4-8 weeks. Nonetheless, it has potent efficacy in patientswith both relapsed/refractory Non-Hodgkin Lymphoma (r/r NHL) and ALL,with step-up dosing (SUD) required to mitigate severe cytokine releasesyndrome and CNS toxicities (Nagorsen and Baeuerle, Exp Cell Res (2011)317, 1255-1260).

The CD20 CD3 targeting T cell bispecific molecule, CD20×CD3 bsAB, isanother example of a next generation of B cell targeting antibody.CD20×CD3 bsAB is a T cell bispecific (TCB) antibody targeting CD20expressed on B cells and CD3 epsilon chain (CD3e) present on T cells.

The mechanism of action of CD20×CD3 bsAB comprises simultaneous bindingto CD20⁺ B cells and CD3⁺ T cells, leading to T-cell activation andT-cell mediated killing of B cells. In the presence of CD20⁺ B cells,whether circulating or tissue resident, pharmacologically active doseswill trigger T-cell activation and associated cytokine release. CD20×CD3bsAB has shown enhanced potency in nonclinical models over competitive Tcell engaging agents and, having an IgG-based format, has a greatlyimproved half-life over blinatumomab.

Cytokine release is the result of activation of T cells. In a phase 1study conducted by TeGenero (Suntharalingam et al., N Engl J Med (2006)355, 1018-1028), all 6 healthy volunteers experienced near fatal, severecytokine release syndrome (CRS) rapidly post-infusion of aninappropriately-dosed, T-cell stimulating super-agonist anti-CD28monoclonal antibody. More recently, in the above-mentioned study byMaude et al. of CD19-targeting, chimeric antigen receptor T cell (CAR-Tcell) treatment of patients with relapsed ALL, all 30 patients hadcytokine release, which was categorized as severe in 27% of thepatients. CRS is a common but severe complication of CAR-T cell therapy(reviewed in Xu and Tang, Cancer Letters (2014) 343, 172-178).

Severe CRS and CNS toxicity have also been frequently observed with theCD19-CD3 T cell bispecific agent, blinatumomab (Klinger et al., Blood.2012; 119(26):6226-6233). In patients receiving blinatumomab in allclinical trials, neurological toxicities have occurred in approximately50% of patients, and the types of toxicities observed are well-definedin the package insert.

It is not well understood if or how CNS toxicity is related to earliercytokine release or T cell activation. Similar to blinatumomab, CNS AEs(ranging from delirium to global encephalopathy) were reported for 43%(13/30) of the patients with r/r ALL treated with CD19-targeting CAR-Tcells (Maude et al., N Engl J Med (2014) 371, 1507-1517; Ghorashian etal., Br J Haematol (2015) 169, 463-478). Neurologic toxic effectstypically occurred after symptoms of CRS had peaked and started toresolve; however no direct, unequivocal association with severe CRS wasfound. The authors proposed that the mechanism of neurotoxicity couldinvolve direct CAR-T-cell-mediated toxicity or it could becytokine-mediated. In contrast, an association between severe CRS andneurotoxicity (e.g., encephalopathy) has been suggested in another studyof CD19-targeting CAR-T cell therapy (Davila et al., Sci Transl Med(2014) 6, 224ra25) and speculated to be due to general T cellactivation, versus direct CAR-T-induced damage.

Cytokine release and/or CNS-related toxicities are particularlypronounced in T cell bispecific antibodies that link CD3⁺ cells to Bcells, as compared to other T cell bispecific antibodies that link CD3⁺cells to tissue-restricted (i.e., non-circulating) target cells. Thereis thus a need for methods to reduce or prevent such adverse effects ofthese promising agents which have the potential to significantlycontribute to the treatment of patients with B-cell proliferativedisorders such as NHL and CLL.

SUMMARY OF THE INVENTION

The present invention is based on the surprising finding that (i) theformation of ADAs in response to administration of an immunogenictherapeutic agent to a subject can effectively and sustainably beprevented, and (ii) the cytokine release associated with administrationof a therapeutic agent, particularly a T-cell activating therapeuticagent such as CD20×CD3 bsAB, to a subject can be significantly reduced,by pre-treatment of said subject with a Type II anti-CD20 antibody, suchas obinutuzumab.

Obinutuzumab is a humanized glyco-engineered type II anti-CD20 mAb thatbinds with high-affinity to the CD20 antigen, inducingantibody-dependent cellular cytotoxicity (ADCC) and antibody-dependentcellular phagocytosis (ADCP), low complement-dependent cytotoxicity(CDC) activity, and high direct cell death induction. To date, thesafety profile of obinutuzumab (including cytokine release) has beenassessed and managed in hundreds of patients in ongoing obinutuzumabclinical trials.

Without wishing to be bound by theory, the use of obinutuzumab (GAZYVA®)pre-treatment (GPT) should aid in the rapid depletion of B cells, bothin the peripheral blood and in secondary lymphoid organs, such that therisk of highly relevant adverse events (AEs) from strong systemic T cellactivation by (T-cell activating) therapeutic agents (e.g. CRS) isreduced, while supporting exposure levels of therapeutic agents that arehigh enough from the start of dosing to mediate tumour cell elimination.In addition to supporting the safety profile of (T-cell activating)therapeutic agents such as CD20×CD3 bsAB, GPT should also help preventthe formation of anti-drug antibodies (ADAs) to therapeutic molecules.

For patients, GPT should translate into better drug exposure with anenhanced safety profile.

GPT should be more effective in accomplishing the above goals comparedto other methods used, such as step up dosing (SUD). For example, asingle dose of obinutuzumab should allow relapsed/refractory patients toreceive the full therapeutic dose of T-cell activating therapeutic agentsuch as CD20×CD3 bsAB, once determined, without a time delay from stepup dosing. In contrast thereto, it was recently reported that theblinatumomab dosing regimen for patients with r/r DLBCL in an ongoingPhase 2 trial incorporates a double step up approach (i.e., 9→28→112μg/m²/day), thus, requiring 14 days to reach the maximum dose of 112μg/m²/day (Viardot el at., Hematol Oncol (2015) 33, 242 (Abstract 285)).As shown in the Examples, following pretreatment with obinutuzumab,administration of CD20×CD3 bsAB to cynomolgus monkeys was tolerated upto a level that was ten times higher than that tolerated without GPT.Efficient peripheral blood B-cell depletion and anti-tumour activityalong with strongly reduced cytokine release in the peripheral bloodassociated with the first CD20×CD3 bsAB injection was observed upon GPT.

Accordingly, in a first aspect the present invention provides a methodfor (i) reducing the formation of anti-drug antibodies (ADAs) against atherapeutic agent in a subject and/or (ii) reducing cytokine releaseassociated with administration of a therapeutic agent, particularly aT-cell activating therapeutic agent, in a subject, comprisingadministration of a Type II anti-CD20 antibody to the subject prior toadministration of the therapeutic agent. In one embodiment the period oftime between the administration of the Type II anti-CD20 antibody andadministration of the therapeutic agent is sufficient for reduction ofthe number of B-cells in the subject in response to the administrationof the Type II anti-CD20 antibody.

In a further aspect, the invention provides a method of treating adisease in a subject, the method comprising a treatment regimencomprising

-   -   (i) administration to the subject of a Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (ii) administration to the subject of a therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent is sufficient for reduction of the number of        B-cells in the subject in response to the administration of the        Type II anti-CD20 antibody.

In one embodiment, the treatment regimen effectively reduces theformation of anti-drug antibodies (ADAs) in the subject in response tothe administration of the therapeutic agent as compared to acorresponding treatment regimen without the administration of the TypeII anti-CD20 antibody.

In another embodiment, the treatment regimen effectively reducescytokine release associated with the administration of the therapeuticagent in the subject as compared to a corresponding treatment regimenwithout the administration of the Type II anti-CD20 antibody. In suchembodiment, the therapeutic agent preferably is a T cell activatingtherapeutic agent.

In a further aspect, the invention provides a Type II anti-CD20 antibodyfor use in a method for (i) reducing the formation of anti-drugantibodies (ADAs) against a therapeutic agent in a subject and/or (ii)reducing cytokine release associated with the administration atherapeutic agent, particularly a T-cell activating therapeutic agent,in a subject, comprising administration of the Type II anti-CD20antibody to the subject prior to administration of the therapeuticagent.

In one embodiment, the period of time between the administration of theType II anti-CD20 antibody and administration of the therapeutic agentis sufficient for reduction of the number of B-cells in the subject inresponse to the administration of the CD20 antibody.

In a further aspect, the invention provides a Type II anti-CD20 antibodyfor use in a method of treating a disease in a subject, the methodcomprising a treatment regimen comprising

-   -   (i) administration to the subject of the Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (ii) administration to the subject of a therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent is sufficient for reduction of the number of        B-cells in the subject in response to the administration of the        Type II anti-CD20 antibody.

In one embodiment, the treatment regimen effectively reduces theformation of anti-drug antibodies (ADAs) against the therapeutic agentin the subject (in response to the administration of the therapeuticagent) as compared to a corresponding treatment regimen without theadministration of the anti-CD20 antibody.

In another embodiment, the treatment regimen effectively reducescytokine release associated with the administration of the therapeuticagent in the subject as compared to a corresponding treatment regimenwithout the administration of the Type II anti-CD20 antibody. In suchembodiment, the therapeutic agent preferably is a T cell activatingtherapeutic agent.

In a further aspect, the invention provides the use of a Type IIanti-CD20 antibody in the manufacture of a medicament for (i) reductionof the formation of anti-drug antibodies (ADAs) against a therapeuticagent in a subject and/or (ii) the reduction of cytokine releaseassociated with administration of a therapeutic agent, particularly aT-cell activating therapeutic agent, in a subject, wherein themedicament is to be used in a treatment regimen comprising

-   -   (i) administration to the subject of the Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (ii) administration to the subject of a therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent is sufficient for reduction of the number of        B-cells in the subject in response to the administration of the        Type II anti-CD20 antibody.

In one embodiment, the treatment regimen effectively reduces theformation of anti-drug antibodies (ADAs) against the therapeutic agentin the subject as compared to a corresponding treatment regimen withoutthe administration of the anti-CD20 antibody.

In another embodiment, the treatment regimen effectively reducescytokine release associated with administration of the therapeutic agentin the subject as compared to a corresponding treatment regimen withoutthe administration of the Type II anti-CD20 antibody. In suchembodiment, the therapeutic agent preferably is a T cell activatingtherapeutic agent.

In still a further aspect, the invention provides a kit for (i) thereduction of the formation of anti-drug antibodies (ADAs) against atherapeutic agent in a subject and/or (ii) the reduction of cytokinerelease associated with administration of a therapeutic agent,particularly a T-cell activating therapeutic agent, in a subject,comprising a package comprising a Type II anti-CD20 antibody compositionand instructions for using the Type II anti-CD20 antibody composition ina treatment regimen comprising

-   -   (i) administration to the subject of the Type II anti-CD20        antibody composition,    -   and consecutively after a period of time    -   (ii) administration to the subject of a therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody composition and the administration of        the therapeutic agent is sufficient for reduction of the number        of B-cells in the subject in response to the administration of        the Type II CD20 antibody.

In one embodiment, the treatment regimen effectively reduces theformation of anti-drug antibodies (ADAs) against the therapeutic agentin the subject as compared to a corresponding treatment regimen withoutthe administration of the Type II anti-CD20 antibody composition.

In another embodiment, the treatment regimen effectively reducescytokine release associated with administration of the therapeutic agentin the subject as compared to a corresponding treatment regimen withoutthe administration of the Type II anti-CD20 antibody composition. Insuch embodiment, the therapeutic agent preferably is a T cell activatingtherapeutic agent. In one embodiment, the kit further comprises atherapeutic agent composition.

The invention in a further aspect as provides a therapeutic agent foruse in a method of treating a disease in a subject, the methodcomprising a treatment regimen comprising

-   -   (i) administration to the subject of a Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (ii) administration to the subject of the therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent is sufficient for reduction of the number of        B-cells in the subject in response to the administration of the        CD20 antibody.

In one embodiment, the treatment regimen effectively reduces theformation of anti-drug antibodies (ADAs) in the subject in response tothe administration of the therapeutic agent as compared to acorresponding treatment regimen without the administration of the TypeII anti-CD20 antibody.

In another embodiment, the treatment regimen effectively reducescytokine release associated with administration of the therapeutic agentin the subject as compared to a corresponding treatment regimen withoutthe administration of the Type II anti-CD20 antibody. In suchembodiment, the therapeutic agent preferably is a T cell activatingtherapeutic agent.

The invention in still a further aspect provides the use of atherapeutic agent in the manufacture of a medicament for treatment of adisease in a subject, wherein the treatment comprises a treatmentregimen comprising

-   -   (i) administration to the subject of a Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (ii) administration to the subject of the therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent is sufficient for reduction of the number of        B-cells in the subject in response to the administration of the        Type II anti-CD20 antibody.

In one embodiment, the treatment regimen effectively reduces theformation of anti-drug antibodies (ADAs) in the subject in response tothe administration of the therapeutic agent as compared to acorresponding treatment regimen without the administration of the TypeII anti-CD20 antibody.

In another embodiment, the treatment regimen effectively reducescytokine release associated with administration of the therapeutic agentin the subject as compared to a corresponding treatment regimen withoutthe administration of the Type II anti-CD20 antibody. In suchembodiment, the therapeutic agent preferably is a T cell activatingtherapeutic agent.

The invention in a further aspect provides a kit for the treatment of adisease in a subject, comprising a package comprising a therapeuticagent composition and instructions for using the therapeutic agentcomposition in a treatment regimen comprising

-   -   (i) administration to the subject of a Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (ii) administration to the subject of the therapeutic agent        composition,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent composition is sufficient for reduction of the        number of B-cells in the subject in response to the        administration of the Type II anti-CD20 antibody.

In one embodiment, the treatment regimen effectively reduces theformation of anti-drug antibodies (ADAs) against the therapeutic agentin the subject as compared to a corresponding treatment regimen withoutthe administration of the Type II anti-CD20 antibody composition.

In another embodiment, the treatment regimen effectively reducescytokine release associated with administration of the therapeutic agentin the subject as compared to a corresponding treatment regimen withoutthe administration of the Type II anti-CD20 antibody composition. Insuch embodiment, the therapeutic agent preferably is a T cell activatingtherapeutic agent. In one embodiment, the kit further comprises a TypeII anti-CD20 antibody composition.

The methods, uses, Type II anti-CD20 antibodies, therapeutic agents andkits of the invention may incorporate, singly or in combination, any ofthe features described hereinbelow.

In one embodiment, the Type II anti-CD20 antibody comprises a heavychain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ IDNO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and alight chain variable region comprising the light chain CDR (LCDR) 1 ofSEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.

In a more specific embodiment, the Type II anti-CD20 antibody comprisesthe heavy chain variable region sequence of SEQ ID NO: 10 and the lightchain variable region sequence of SEQ ID NO: 11.

In one embodiment, the Type II anti-CD20 antibody is an IgG antibody,particularly an IgG₁ antibody.

In one embodiment, the Type II anti-CD20 antibody is engineered to havean increased proportion of non-fucosylated oligosaccharides in the Fcregion as compared to a non-engineered antibody. In one embodiment, atleast about 40% of the N-linked oligosaccharides in the Fc region of theType II anti-CD20 antibody are non-fucosylated.

In a particular embodiment the anti-CD20 antibody is obinutuzumab.

In some embodiments, in particular in relation aspects of the inventionconcerned with the reduction of the formation of anti-drug antibodies(ADAs) against a therapeutic agent in a subject, the therapeutic agentcomprises a polypeptide.

In some embodiments, in particular in relation aspects of the inventionconcerned with the reduction of the formation of anti-drug antibodies(ADAs) against a therapeutic agent in a subject, the therapeutic agentcomprises an antibody.

In one such embodiment, the antibody specifically binds tocarcinoembryonic antigen (CEA). In one embodiment, the antibodycomprises a heavy chain variable region comprising the heavy chain CDR(HCDR) 1 of SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15, and the HCDR3 ofSEQ ID NO: 16; and a light chain variable region comprising the lightchain CDR (LCDR) 1 of SEQ ID NO: 17, the LCDR2 of SEQ ID NO: 18 and theLCDR3 of SEQ ID NO: 19. In a further embodiment, the antibody comprisesthe heavy chain variable region sequence of SEQ ID NO: 20 and the lightchain variable region sequence of SEQ ID NO: 21. In another suchembodiment, the antibody specifically binds to CD3, particularly CD3epsilon. In one embodiment, the antibody comprises a heavy chainvariable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO:32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and alight chain variable region comprising the light chain CDR (LCDR) 1 ofSEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO:37. In a further embodiment, the antibody comprises the heavy chainvariable region sequence of SEQ ID NO: 38 and the light chain variableregion sequence of SEQ ID NO: 39.

In some embodiments, in particular in relation aspects of the inventionconcerned with the reduction of the formation of anti-drug antibodies(ADAs) against a therapeutic agent in a subject, the therapeutic agentcomprises a cytokine.

In one such embodiment, the cytokine is interleukin-2 (IL-2).

In another such embodiment, the cytokine is a mutant human IL-2polypeptide comprising the amino acid substitutions F42A, Y45A and L72G(numbering relative to the human IL-2 sequence SEQ ID NO: 12).

In some embodiments, in particular in relation aspects of the inventionconcerned with the reduction of the formation of anti-drug antibodies(ADAs) against a therapeutic agent in a subject, the therapeutic agentcomprises an immunoconjugate.

In one such embodiment, the immunoconjugate comprises (a) an antibodythat specifically binds to CEA and comprises a heavy chain variableregion comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 14, theHCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO: 16; and a lightchain variable region comprising the light chain CDR (LCDR) 1 of SEQ IDNO: 17, the LCDR2 of SEQ ID NO: 18 and the LCDR3 of SEQ ID NO: 19, and(b) a mutant human IL-2 polypeptide comprising the amino acidsubstitutions F42A, Y45A and L72G (numbering relative to the human IL-2sequence SEQ ID NO: 12).

In a particular such embodiment, the therapeutic agent comprisescergutuzumab amunaleukin (CEA-IL2v).

In some embodiments, in particular in relation aspects of the inventionconcerned with the reduction of the formation of anti-drug antibodies(ADAs) against a therapeutic agent in a subject, the therapeutic agentcomprises a bispecific antibody that specifically binds to CEA and toCD3.

In one such embodiment the therapeutic agent comprises a bispecificantibody comprising

-   -   (i) an antigen binding moiety that specifically binds to CD3 and        comprises a heavy chain variable region comprising the heavy        chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33,        and the HCDR3 of SEQ ID NO: 34; and a light chain variable        region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35,        the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37; and    -   (ii) an antigen binding moiety that specifically bind to CEA and        comprises a heavy chain variable region comprising the heavy        chain CDR (HCDR) 1 of SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15,        and the HCDR3 of SEQ ID NO: 16; and a light chain variable        region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 17,        the LCDR2 of SEQ ID NO: 18 and the LCDR3 of SEQ ID NO: 19.

In a particular embodiment, the therapeutic agent comprises CEA TCB.

In some embodiments, in particular in relation aspects of the inventionconcerned with the reduction of cytokine release associated with theadministration of a therapeutic agent in a subject, the therapeuticagent is a T cell activating therapeutic agent.

In one embodiment, the T-cell activating therapeutic agent comprises anantibody, particularly a multispecific (e.g. a bispecific) antibody.

In one embodiment, the antibody specifically binds to an activating Tcell antigen.

In one embodiment, the antibody specifically binds to an antigenselected from the group of CD3, CD28, CD137 (also known as 4-1BB), CD40,CD226, OX40, GITR, CD27, HVEM, and CD127.

In one embodiment, the antibody specifically binds to CD3, particularlyCDR.

In one embodiment, the antibody comprises a heavy chain variable regioncomprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 ofSEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chainvariable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO:35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37.

In one embodiment, the antibody comprises the heavy chain variableregion sequence of SEQ ID NO: 38 and the light chain variable regionsequence of SEQ ID NO: 39.

In one embodiment, the antibody specifically binds to a B-cell antigen,particularly a malignant B-cell antigen.

In one embodiment, the antibody specifically binds to an antigenselected from the group consisting of CD20, CD19, CD22, ROR-1, CD37 andCD5, particularly to CD20 or CD19.

In one embodiment, the antibody specifically binds to CD20.

In one embodiment, the antibody comprises a heavy chain variable regioncomprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 ofSEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light chain variableregion comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, theLCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.

In one embodiment, the antibody comprises the heavy chain variableregion sequence of SEQ ID NO: 10 and the light chain variable regionsequence of SEQ ID NO: 11.

In one embodiment, the antibody is a multispecific antibody,particularly a bispecific antibody.

In one embodiment, the multispecific antibody specifically binds to (i)an activating T cell antigen and (ii) a B cell antigen.

In one embodiment, the multispecific antibody specifically binds to (i)CD3 and (ii) an antigen selected from CD20 and CD19.

In one embodiment, the multispecific antibody specifically binds to CD3and CD20.

In some embodiments, in particular in relation aspects of the inventionconcerned with the reduction of cytokine release associated with theadministration of a therapeutic agent in a subject, the therapeuticagent comprises a bispecific antibody comprising

-   -   (i) an antigen binding moiety that specifically binds to CD3 and        comprises a heavy chain variable region comprising the heavy        chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33,        and the HCDR3 of SEQ ID NO: 34; and a light chain variable        region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35,        the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37; and    -   (ii) an antigen binding moiety that specifically binds to CD20        and comprises a heavy chain variable region comprising the heavy        chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5,        and the HCDR3 of SEQ ID NO: 6; and a light chain variable region        comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the        LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.

In a particular embodiment, the therapeutic agent comprises CD20×CD3bsAB.

In some embodiments, in particular in relation aspects of the inventionconcerned with the reduction of cytokine release associated with theadministration of a therapeutic agent in a subject, the therapeuticagent comprises a chimeric antigen receptor (CAR) or a T cell expressinga CAR, particularly a CAR that specifically binds to a B-cell antigen,more particularly a CAR that specifically binds to an antigen selectedfrom the group of CD20, CD19, CD22, ROR-1, CD37 and CD5.

In some embodiments, in particular in relation aspects of the inventionconcerned with the reduction of cytokine release associated with theadministration of a therapeutic agent in a subject, the disease is a Bcell proliferative disorder, particularly a CD20-positive B-celldisorder. In one embodiment, the disease is selected from the groupconsisting of Non-Hodgkin lymphoma (NHL), acute lymphocytic leukemia(ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma(DLBCL), follicular lymphoma (FL), mantle-cell lymphoma (MCL), marginalzone lymphoma (MZL), Multiple myeloma (MM), and Hodgkin lymphoma (HL).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Prior treatment with obinutuzumab but not rituximab or vehicleresults in the attenuation of tetanus toxoid specific de novo IgGantibody responses in cynomolgus monkeys. Rituxan indicates rituximaband GA101 obinutuzumab, respectively.

FIG. 2 . Memory recall responses by measles specific IgG antibodyproduction in response to immune re-challenge with a measles/rubellabooster vaccination in animals with baseline titers to measles is notaffected by either obinutuzumab or rituximab in cynomolgus monkeys.Rituxan indicates rituximab and GA101 obinutuzumab, respectively.

FIG. 3 . B cell counts (CD45+CD19+) in peripheral blood samples beforestart of obinutuzumab pre-treatment (BL=baseline), before start oftreatment with RO6895882 (C1D1=Cycle 1 Day 1) and during treatment withRO6895882. Lines/symbols represent individual patients. From the C1D1time points onwards, no B cells were detectable in the peripheral bloodsamples.

FIGS. 4A-C. Reduction of CD19+ cells (B cells) detected by flowcytometry in tumor biopsies collected at baseline (BL) and aftertreatment with obinutuzumab (treated). On-treatment samples wereobtained either before or during treatment with RO6895882. Thepercentage of CD45+ cells (lymphocytes) staining positive for CD19 (Blymphocytes) was strongly reduced (B). No clear change was observed forthe percentage of CD16+ cells (Natural Killer Cells) (A) or CD3+ cells(T lymphocytes) (C). Lines represent individual patients.

FIGS. 5A-D. Reduction of B cells in tumor biopsies collected at baseline(BL) and after treatment (treated) with obinutuzumab measured byimmunohistochemistry. On-treatment samples were obtained either beforeor during treatment with RO6895882. The density of B lymphocytes wasmeasured by staining with CD20 (A, B) and PAX 5 (C, D). Both methodsdetected a depletion of B lymphocytes in tumor and surrounding normaltissue. Lines represent individual patients.

FIGS. 6A-Z. Exemplary configurations of the T cell activating bispecificantigen binding molecules (TCBs) useful in the invention. (A, D)Illustration of the “1+1 CrossMab” molecule. (B, E) Illustration of the“2+1 IgG Crossfab” molecule with alternative order of Crossfab and Fabcomponents (“inverted”). (C, F) Illustration of the “2+1 IgG Crossfab”molecule. (G, K) Illustration of the “1+1 IgG Crossfab” molecule withalternative order of Crossfab and Fab components (“inverted”). (H, L)Illustration of the “1+1 IgG Crossfab” molecule. (I, M) Illustration ofthe “2+1 IgG Crossfab” molecule with two CrossFabs. (J, N) Illustrationof the “2+1 IgG Crossfab” molecule with two CrossFabs and alternativeorder of Crossfab and Fab components (“inverted”). (O, S) Illustrationof the “Fab-Crossfab” molecule. (P, T) Illustration of the“Crossfab-Fab” molecule. (Q, U) Illustration of the “(Fab)₂-Crossfab”molecule. (R, V) Illustration of the “Crossfab-(Fab)₂” molecule. (W, Y)Illustration of the “Fab-(Crossfab)₂” molecule. (X, Z) Illustration ofthe “(Crossfab)₂-Fab” molecule. Black dot: optional modification in theFc domain promoting heterodimerization. ++, −−: amino acids of oppositecharges optionally introduced in the CH1 and CL domains. Crossfabmolecules are depicted as comprising an exchange of VH and VL regions,but may—in embodiments wherein no charge modifications are introduced inCH1 and CL domains—alternatively comprise an exchange of the CH1 and CLdomains.

FIGS. 7A-B. B cell and T cell counts in the peripheral blood in thedifferent treatment groups. Flow cytometry analysis of CD19⁺ B cells (A)and CD3⁺ T cells (B) in the peripheral blood of vehicle and CD20×CD3bsAB-treated fully humanized NOG mice, 24 hours and 72 hours after firstand second CD20×CD3 bsAB administration. Black arrows indicate days ofCD20×CD3 bsAB administration.

FIGS. 8A-C. Cytokines released in peripheral blood among the differenttreatment groups. Multiplex analysis of cytokines in blood of vehicleand treated mice, 24 hours and 72 hours after the first and secondadministration of CD20×CD3 bsAB. Histogram bars represent the mean of 5animals with error bars indicating the standard deviation.Representative graphs for IFNγ (A), TNFα (B) and IL-6 (C) are shown.Compare the cytokine release of the first injection of CD20×CD3 bsABwith and without obinutuzumab pre-treatment (bars to be compared areindicated by connecting lines).

FIG. 9 . Anti-tumour activity of CD20×CD3 bsAB, obinutuzumab, andobinutuzumab pretreatment (Gpt)+CD20×CD3 bsAB. Anti-tumour activity ofCD20×CD3 bsAB and obinutuzumab as monotherapy or Gpt+CD20×CD3 bsAB infully humanized NOG mice. Black arrow indicates start of therapy.(8<n<10). Tumour model: WSU-DLCL2.

FIGS. 10A-E. Cytokines released in peripheral blood of cynomolgusmonkeys following dosing with CD20×CD3 bsAB and Gpt+CD20×CD3 bsABtreatments. (A) IFNγ, (B) IL-8, (C) TNFα, (D) IL-2, (E) IL-6.

FIG. 11 . Anti-tumor activity upon step-up dosing of CD20×CD3 bsAB andobinutuzumab pretreatment (Gpt) in fully humanized NOG mice bearingWSU-DLCL2 tumors. Mice received a first therapy (arrow) either as afractionated dose of CD20×CD3 bsAB (0.15, 0.05, 0.015 mg/kg IV) or Gpt(10 mg/kg obinutuzumab), followed by weekly intravenous injections ofCD20×CD3 bsAB at 0.5 mg/kg (full dose) for 9 treatment cycles (i.e., 9weeks). In the vehicle group, one single mouse is shown from day 18. Forthe other groups, n=9 or 10. Tumor model: WSU-DLCL2 injectedsubcutaneously. [CD20×CD3 bsAB 0.05 mg/kg+CD20×CD3 bsAB 0.05 mg/kg] vs[obinutuzumab 10 mg/kg+CD20×CD3 bsAB 0.5 mg/kg]*p=0.018 (One-way ANOVAanalysis of sAUC with Dunnet's method).

FIGS. 12A-H. T-cell staining in lungs from fully humanized NOG micebearing WSU-DLCL2 tumors, (A-D) 7 days after the first treatment, and(E-H) 24 hours after the second treatment. Treatment groups are asfollows (single treatment or first+second treatment): (A) vehicle, (B)obinutuzumab 10 mg/kg, (C) CD20×CD3 bsAB 0.15 mg/kg, (D) CD20×CD3 bsAB0.5 mg/kg, (E) vehicle+vehicle, (F) obinutuzumab 10 mg/kg+CD20×CD3 bsAB0.5 mg/kg, (G) CD20×CD3 bsAB 0.15 mg/kg+CD20×CD3 bsAB 0.5 mg/kg, (H)vehicle+CD20×CD3 bsAB 0.5 mg/kg. Lung sections areimmunohistochemically-stained with anti-CD3 antibody (dark); nuclei werecounterstained with hematoxylin. Magnification 20×. Arrows point toincrease in perivascular CD3 positive cells.

FIGS. 13A-B. Lung of humanized NOG mouse sacrificed 24 hours aftersingle treatment with 0.5 mg/kg of CD20×CD3 bsAB. Margination andadhesion of T cells (arrows) to the endothelium in vessels. Few T cellshave transmigrated to the perivascular space (asterisks). (A) 20×magnification, (B) 40× magnification.

FIG. 14 . Serum concentration-time curves of CD20×CD3 bsAB following asingle intravenous administration with or without obinutuzumabPretreatment in cynomolgus monkeys. Cynomolgus monkeys were administereda single IV dose of 100-1000 μg/kg CD20×CD3 bsAB with or withoutobinutuzumab pretreatment (Gpt) (50 mg/kg, 4 days prior to CD20×CD3 bsABadministration). Six animals are represented in each dose group and thedata is presented as mean±SD.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms are used herein as generally used in the art, unless otherwisedefined in the following.

CD20 (also known as B-lymphocyte antigen CD20, B-lymphocyte surfaceantigen B1, Leu-16, Bp35, BMS, and LF5; the human protein ischaracterized in UniProt database entry P11836) is a hydrophobictransmembrane protein with a molecular weight of approximately 35 kDexpressed on pre-B and mature B lymphocytes (Valentine, M. A. et al., J.Biol. Chem. 264 (1989) 11282-11287; Tedder, T. F., et al., Proc. Natl.Acad. Sci. U.S.A. 85 (1988) 208-212; Stamenkovic, I., et al., J. Exp.Med. 167 (1988) 1975-1980; Einfeld, D. A., et al., EMBO J. 7 (1988)711-717; Tedder, T. F., et al., J. Immunol. 142 (1989) 2560-2568). Thecorresponding human gene is Membrane-spanning 4-domains, subfamily A,member 1, also known as MS4A1. This gene encodes a member of themembrane-spanning 4A gene family. Members of this nascent protein familyare characterized by common structural features and similar intron/exonsplice boundaries and display unique expression patterns amonghematopoietic cells and nonlymphoid tissues. This gene encodes theB-lymphocyte surface molecule which plays a role in the development anddifferentiation of B-cells into plasma cells. This family member islocalized to 11q12, among a cluster of family members. Alternativesplicing of this gene results in two transcript variants which encodethe same protein.

The term “CD20” as used herein, refers to any native CD20 from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed CD20 as well as any form of CD20that results from processing in the cell. The term also encompassesnaturally occurring variants of CD20, e.g., splice variants or allelicvariants. In one embodiment, CD20 is human CD20. The amino acid sequenceof an exemplary human CD20 is shown in SEQ ID NO: 1.

The terms “anti-CD20 antibody” and “an antibody that binds to CD20”refer to an antibody that is capable of binding CD20 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting CD20. In one embodiment, the extent ofbinding of an anti-CD20 antibody to an unrelated, non-CD20 protein isless than about 10% of the binding of the antibody to CD20 as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to CD20 has a dissociation constant (Kd) of ≤1 μM, ≤100 nM,≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less,e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certainembodiments, an anti-CD20 antibody binds to an epitope of CD20 that isconserved among CD20 from different species.

By “Type II anti-CD20 antibody” is meant an anti-CD20 antibody havingbinding properties and biological activities of Type II anti-CD20antibodies as described in Cragg et al., Blood 103 (2004) 2738-2743;Cragg et al., Blood 101 (2003) 1045-1052, Klein et al., mAbs 5 (2013),22-33, and summarized in Table 1 below.

TABLE 1 Properties of type I and type II anti-CD20 antibodies type Ianti-CD20 antibodies type II anti-CD20 antibodies Bind class I CD20epitope Bind class II CD20 epitope Localize CD20 to lipid rafts Do notlocalize CD20 to lipid rafts High CDC * Low CDC * ADCC activity * ADCCactivity * Full binding capacity to B cells Approx. half bindingcapacity to B cells Weak homotypic aggregation Homotypic aggregation Lowcell death induction Strong cell death induction * if IgG₁ isotype

Examples of type II anti-CD20 antibodies include e.g. obinutuzumab(GA101), tositumomab (B1), humanized B-Ly1 antibody IgG1 (a chimerichumanized IgG1 antibody as disclosed in WO 2005/044859), 11B8 IgG1 (asdisclosed in WO 2004/035607) and AT80 IgG1.

Examples of type I anti-CD20 antibodies include e.g. rituximab,ofatumumab, veltuzumab, ocaratuzumab, ocrelizumab, PRO131921,ublituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (as disclosed in WO2005/103081), 2F2 IgG1 (as disclosed in WO 2004/035607 and WO2005/103081) and 2H7 IgG1 (as disclosed in WO 2004/056312).

The term “humanized B-Ly1 antibody” refers to humanized B-Ly1 antibodyas disclosed in WO 2005/044859 and WO 2007/031875, which were obtainedfrom the murine monoclonal anti-CD20 antibody B-Ly1 (variable region ofthe murine heavy chain (VH): SEQ ID NO: 2; variable region of the murinelight chain (VL): SEQ ID NO: 3 (see Poppema, S. and Visser, L., BiotestBulletin 3 (1987) 131-139) by chimerization with a human constant domainfrom IgG1 and following humanization (see WO 2005/044859 and WO2007/031875). These “humanized B-Ly1 antibodies” are disclosed in detailin WO 2005/044859 and WO 2007/031875.

As used herein, the term “cytokine” refers to a molecule that mediatesand/or regulates a biological or cellular function or process (e.g.immunity, inflammation, and hematopoiesis). The term “cytokine” as usedherein includes “lymphokines,” “chemokines,” “monokines,” and“interleukins”. Examples of useful cytokines include, but are notlimited to, GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-12, IL-15, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β,TNF-α, and TNF-β. A particular cytokines is IL-2. The term “cytokine” asused herein is meant to also include cytokine variants comprising one ormore amino acid mutations in the amino acid sequences of thecorresponding wild-type cytokine, such as for example the IL-2 variantsdescribed in Sauvé et al., Proc Natl Acad Sci USA 88, 4636-40 (1991); Huet al., Blood 101, 4853-4861 (2003) and US Pat. Publ. No. 2003/0124678;Shanafelt et al., Nature Biotechnol 18, 1197-1202 (2000); Heaton et al.,Cancer Res 53, 2597-602 (1993) and U.S. Pat. No. 5,229,109; US Pat.Publ. No. 2007/0036752; WO 2008/0034473; WO 2009/061853; or in WO2012/107417.

The term “interleukin-2” or “IL-2” as used herein, refers to any nativeIL-2 from any vertebrate source, including mammals such as primates(e.g. humans) and rodents (e.g., mice and rats), unless otherwiseindicated. The term encompasses unprocessed IL-2 as well as any form ofIL-2 that results from processing in the cell. The term also encompassesnaturally occurring variants of IL-2, e.g. splice variants or allelicvariants. The amino acid sequence of an exemplary human IL-2 is shown inSEQ ID NO: 12. Unprocessed human IL-2 additionally comprises anN-terminal 20 amino acid signal peptide having the sequence of SEQ IDNO: 31, which is absent in the mature IL-2 molecule. The term“interleukin-2” as used herein is meant to also include IL-2 variantscomprising one or more amino acid mutations in the amino acid sequencesof the corresponding wild-type cytokine, such as for example the IL-2variants described in Sauvé et al., Proc Natl Acad Sci USA 88, 4636-40(1991); Hu et al., Blood 101, 4853-4861 (2003) and US Pat. Publ. No.2003/0124678; Shanafelt et al., Nature Biotechnol 18, 1197-1202 (2000);Heaton et al., Cancer Res 53, 2597-602 (1993) and U.S. Pat. No.5,229,109; US Pat. Publ. No. 2007/0036752; WO 2008/0034473; WO2009/061853; or in WO 2012/107417.

The term “IL-2 mutant” or “mutant IL-2 polypeptide” as used herein isintended to encompass any mutant forms of various forms of the IL-2molecule including full-length IL-2, truncated forms of IL-2 and formswhere IL-2 is linked to another molecule such as by fusion or chemicalconjugation. “Full-length” when used in reference to IL-2 is intended tomean the mature, natural length IL-2 molecule. For example, full-lengthhuman IL-2 refers to a molecule that has 133 amino acids (see e.g. SEQID NO: 12). The various forms of IL-2 mutants are characterized inhaving a at least one amino acid mutation affecting the interaction ofIL-2 with CD25. This mutation may involve substitution, deletion,truncation or modification of the wild-type amino acid residue normallylocated at that position. Mutants obtained by amino acid substitutionare preferred. Unless otherwise indicated, an IL-2 mutant may bereferred to herein as an IL-2 mutant peptide sequence, an IL-2 mutantpolypeptide, IL-2 mutant protein or IL-2 mutant analog. Designation ofvarious forms of IL-2 is herein made with respect to the sequence shownin SEQ ID NO: 12. Various designations may be used herein to indicatethe same mutation. For example a mutation from phenylalanine at position42 to alanine can be indicated as 42A, A42, A₄₂, F42A, or Phe42Ala.

As used herein, the term “release of cytokines” or “cytokine release” issynonymous with “cytokine storm” or “cytokine release syndrome”(abbreviated as “CRS”), and refers to an increase in the levels ofcytokines, particularly tumor necrosis factor alpha (TNF-α), interferongamma (IFN-γ), interleukin-6 (IL-6), interleukin-10 (IL-10),interleukin-2 (IL-2) and/or interleukin-8 (IL-8), in the blood of asubject during or shortly after (e.g. within 1 day of) administration ofa therapeutic agent, resulting in adverse symptoms. Cytokine release isa type of infusion-related reaction (IRR), which are common adverse drugreactions to therapeutic agent and timely related to administration ofthe therapeutic agent. IRRs typically occur during or shortly after anadministration of the therapeutic agent, i.e. typically within 24 hoursafter infusion, predominantly at the first infusion. In some instances,e.g. after the administration of CAR-T cells, CRS can also occur onlylater, e.g. several days after administration upon expansion of theCAR-T cells. The incidence and severity typically decrease withsubsequent infusions. Symptoms may range from symptomatic discomfort tofatal events, and may include fever, chills, dizziness, hypertension,hypotension, dyspnea, restlessness, sweating, flushing, skin rash,tachycardia, tachypnea, headache, tumour pain, nausea, vomiting and/ororgan failure.

The term “amino acid mutation” as used herein is meant to encompassamino acid substitutions, deletions, insertions, and modifications. Anycombination of substitution, deletion, insertion, and modification canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., reduced bindingto CD25 or to an Fc receptor. Amino acid sequence deletions andinsertions include amino- and/or carboxy-terminal deletions andinsertions of amino acids. Particular amino acid mutations are aminoacid substitutions. For the purpose of altering e.g. the bindingcharacteristics of an IL-2 polypeptide or an Fc region, non-conservativeamino acid substitutions, i.e. replacing one amino acid with anotheramino acid having different structural and/or chemical properties, areparticularly preferred. Amino acid substitutions include replacement bynon-naturally occurring amino acids or by naturally occurring amino acidderivatives of the twenty standard amino acids (e.g. 4-hydroxyproline,3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acidmutations can be generated using genetic or chemical methods well knownin the art. Genetic methods may include site-directed mutagenesis, PCR,gene synthesis and the like. It is contemplated that methods of alteringthe side chain group of an amino acid by methods other than geneticengineering, such as chemical modification, may also be useful. Variousdesignations may be used herein to indicate the same amino acidmutation. For example, a substitution from proline at position 329 ofthe Fc region to glycine can be indicated as 329G, G329, G₃₂₉, P329G, orPro329Gly.

The term “CD25” or “α-subunit of the IL-2 receptor” as used herein,refers to any native CD25 from any vertebrate source, including mammalssuch as primates (e.g. humans) and rodents (e.g., mice and rats), unlessotherwise indicated. The term encompasses “full-length”, unprocessedCD25 as well as any form of CD25 that results from processing in thecell. The term also encompasses naturally occurring variants of CD25,e.g. splice variants or allelic variants. In certain embodiments CD25 ishuman CD25. The amino acid sequence of human CD25 is shown in UniProt(www.uniprot.org) accession no. P01589, or NCBI (www.ncbi.nlm.nih.gov/)RefSeq NP_000408.

The term “high-affinity IL-2 receptor” as used herein refers to theheterotrimeric form of the IL-2 receptor, consisting of the receptorγ-subunit (also known as common cytokine receptor γ-subunit, γ_(c), orCD132), the receptor β-subunit (also known as CD122 or p′70) and thereceptor α-subunit (also known as CD25 or p55). The term“intermediate-affinity IL-2 receptor” by contrast refers to the IL-2receptor including only the γ-subunit and the β-subunit, without theα-subunit (for a review see e.g. Olejniczak and Kasprzak, Med Sci Monit14, RA179-189 (2008)).

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., areceptor) and its binding partner (e.g., a ligand). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., receptor and a ligand). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (K_(D)), which is the ratio of dissociation and associationrate constants (k_(off) and k_(on), respectively). Thus, equivalentaffinities may comprise different rate constants, as long as the ratioof the rate constants remains the same. Affinity can be measured by wellestablished methods known in the art. A particular method for measuringaffinity is Surface Plasmon Resonance (SPR).

“Reduction” (and grammatical variations thereof such as “reduce” or“reducing”), for example reduction of the number of B cells or theformation of ADAs or cytokine release, refers to a decrease in therespective quantity, as measured by appropriate methods known in theart. For clarity the term includes also reduction to zero (or below thedetection limit of the analytical method), i.e. complete abolishment orelimination. Conversely, “increased” refers to an increase in therespective quantity.

By “regulatory T cell” or “T_(reg) cell” is meant a specialized type ofCD4⁺ T cell that can suppress the responses of other T cells. T_(reg)cells are characterized by expression of the α-subunit of the IL-2receptor (CD25) and the transcription factor forkhead box P3 (FOXP3)(Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)) and play a critical rolein the induction and maintenance of peripheral self-tolerance toantigens, including those expressed by tumors. T_(reg) cells requireIL-2 for their function and development and induction of theirsuppressive characteristics.

As used herein, the term “antigen binding moiety” refers to apolypeptide molecule that specifically binds to an antigenicdeterminant. In one embodiment, an antigen binding moiety is able todirect the entity to which it is attached (e.g. a cytokine or a secondantigen binding moiety) to a target site, for example to a specific typeof tumor cell or tumor stroma bearing the antigenic determinant. Antigenbinding moieties include antibodies and fragments thereof as furtherdefined herein. Preferred antigen binding moieties include an antigenbinding domain of an antibody, comprising an antibody heavy chainvariable region and an antibody light chain variable region. In certainembodiments, the antigen binding moieties may include antibody constantregions as further defined herein and known in the art. Useful heavychain constant regions include any of the five isotypes: α, δ, ε, γ, orμ. Useful light chain constant regions include any of the two isotypes:κ and λ.

By “specifically binds” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antigen binding moiety to bind to aspecific antigenic determinant can be measured either through anenzyme-linked immunosorbent assay (ELISA) or other techniques familiarto one of skill in the art, e.g. surface plasmon resonance technique(analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17,323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28,217-229 (2002)).

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope,” and refers to a site (e.g. a contiguous stretchof amino acids or a conformational configuration made up of differentregions of non-contiguous amino acids) on a polypeptide macromolecule towhich an antigen binding moiety binds, forming an antigen bindingmoiety-antigen complex. Useful antigenic determinants can be found, forexample, on the surfaces of tumor cells, on the surfaces ofvirus-infected cells, on the surfaces of other diseased cells, free inblood serum, and/or in the extracellular matrix (ECM).

As used herein, term “polypeptide” refers to a molecule composed ofmonomers (amino acids) linearly linked by amide bonds (also known aspeptide bonds). The term “polypeptide” refers to any chain of two ormore amino acids, and does not refer to a specific length of theproduct. Thus, peptides, dipeptides, tripeptides, oligopeptides,“protein,” “amino acid chain,” or any other term used to refer to achain of two or more amino acids, are included within the definition of“polypeptide,” and the term “polypeptide” may be used instead of, orinterchangeably with any of these terms. The term “polypeptide” is alsointended to refer to the products of post-expression modifications ofthe polypeptide, including without limitation glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, or modification bynon-naturally occurring amino acids. A polypeptide may be derived from anatural biological source or produced by recombinant technology, but isnot necessarily translated from a designated nucleic acid sequence. Itmay be generated in any manner, including by chemical synthesis. Apolypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded.

By an “isolated” polypeptide or a variant, or derivative thereof isintended a polypeptide that is not in its natural milieu. No particularlevel of purification is required. For example, an isolated polypeptidecan be removed from its native or natural environment. Recombinantlyproduced polypeptides and proteins expressed in host cells areconsidered isolated for the purpose of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco,California, or may be compiled from the source code. The ALIGN-2 programshould be compiled for use on a UNIX operating system, including digitalUNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2program and do not vary. In situations where ALIGN-2 is employed foramino acid sequence comparisons, the % amino acid sequence identity of agiven amino acid sequence A to, with, or against a given amino acidsequence B (which can alternatively be phrased as a given amino acidsequence A that has or comprises a certain % amino acid sequenceidentity to, with, or against a given amino acid sequence B) iscalculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

As used herein, the term “effector moiety” refers to a polypeptide,e.g., a protein or glycoprotein, that influences cellular activity, forexample, through signal transduction or other cellular pathways.Accordingly, the effector moiety can be associated withreceptor-mediated signaling that transmits a signal from outside thecell membrane to modulate a response in a cell bearing one or morereceptors for the effector moiety. In one embodiment, an effector moietycan elicit a cytotoxic response in cells bearing one or more receptorsfor the effector moiety. In another embodiment, an effector moiety canelicit a proliferative response in cells bearing one or more receptorsfor the effector moiety. In another embodiment, an effector moiety canelicit differentiation in cells bearing receptors for the effectormoiety. In another embodiment, an effector moiety can alter expression(i.e. upregulate or downregulate) of an endogenous cellular protein incells bearing receptors for the effector moiety. Non-limiting examplesof effector moieties include cytokines, growth factors, hormones,enzymes, substrates, and cofactors. An effector moiety can be associatedwith an antigen binding moiety such as an antibody in a variety ofconfigurations to form an immunoconjugate.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen binding activity.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂, diabodies, linear antibodies, single-chain antibody molecules(e.g. scFv), and multispecific antibodies formed from antibodyfragments. The term “antibody fragment” as used herein also encompassessingle-domain antibodies.

The term “immunoglobulin molecule” refers to a protein having thestructure of a naturally occurring antibody. For example,immunoglobulins of the IgG class are heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two light chains and two heavy chainsthat are disulfide-bonded. From N- to C-terminus, each heavy chain has avariable region (VH), also called a variable heavy domain or a heavychain variable domain, followed by three constant domains (CH1, CH2, andCH3), also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by aconstant light (CL) domain, also called a light chain constant region.The heavy chain of an immunoglobulin may be assigned to one of fiveclasses, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subclasses, e.g. γ₁ (IgG₁), γ₂ (IgG₂),γ₃ (IgG₃), γ₄ (IgG₄), α₁ (IgA₁) and α₂ (IgA₂). The light chain of animmunoglobulin may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain. Animmunoglobulin essentially consists of two Fab molecules and an Fcdomain, linked via the immunoglobulin hinge region.

The term “antigen binding domain” refers to the part of an antibody thatcomprises the area which specifically binds to and is complementary topart or all of an antigen. An antigen binding domain may be provided by,for example, one or more antibody variable domains (also called antibodyvariable regions). Preferably, an antigen binding domain comprises anantibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindtet al., Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007). A single VH or VL domain may be sufficient to confer antigenbinding specificity.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs: three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

-   -   (a) hypervariable loops occurring at amino acid residues 26-32        (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101        (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));    -   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56        (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3)        (Kabat et al., Sequences of Proteins of Immunological Interest,        5th Ed. Public Health Service, National Institutes of Health,        Bethesda, MD (1991));    -   (c) antigen contacts occurring at amino acid residues 27c-36        (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and        93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745        (1996)); and    -   (d) combinations of (a), (b), and/or (c), including HVR amino        acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2),        26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102        (H3).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an immunoglobulin heavy chain that contains atleast a portion of the constant region. The term includes nativesequence Fc regions and variant Fc regions. Although the boundaries ofthe Fc region of an IgG heavy chain might vary slightly, the human IgGheavy chain Fc region is usually defined to extend from Cys226, or fromPro230, to the carboxyl-terminus of the heavy chain. However, antibodiesproduced by host cells may undergo post-translational cleavage of one ormore, particularly one or two, amino acids from the C-terminus of theheavy chain. Therefore an antibody produced by a host cell by expressionof a specific nucleic acid molecule encoding a full-length heavy chainmay include the full-length heavy chain, or it may include a cleavedvariant of the full-length heavy chain (also referred to herein as a“cleaved variant heavy chain”). This may be the case where the final twoC-terminal amino acids of the heavy chain are glycine (G446) and lysine(K447, numbering according to Kabat EU index). Therefore, the C-terminallysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447),of the Fc region may or may not be present. Unless otherwise specifiedherein, numbering of amino acid residues in the Fc region or constantregion is according to the EU numbering system, also called the EUindex, as described in Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, MD, 1991 (see also above). A “subunit”of an Fc domain as used herein refers to one of the two polypeptidesforming the dimeric Fc domain, i.e. a polypeptide comprising C-terminalconstant regions of an immunoglobulin heavy chain, capable of stableself-association. For example, a subunit of an IgG Fc domain comprisesan IgG CH2 and an IgG CH3 constant domain.

A “modification promoting heterodimerization” is a manipulation of thepeptide backbone or the post-translational modifications of apolypeptide, e.g. an immunoglobulin heavy chain, that reduces orprevents the association of the polypeptide with an identicalpolypeptide to form a homodimer. A modification promotingheterodimerization as used herein particularly includes separatemodifications made to each of two polypeptides desired to form a dimer,wherein the modifications are complementary to each other so as topromote association of the two polypeptides. For example, a modificationpromoting heterodimerization may alter the structure or charge of one orboth of the polypeptides desired to form a dimer so as to make theirassociation sterically or electrostatically favorable, respectively.Heterodimerization occurs between two non-identical polypeptides, suchas two immunoglobulin heavy chains wherein further immunoconjugatecomponents fused to each of the heavy chains (e.g. IL-2 polypeptide) arenot the same. In the immunoconjugates useful in the present invention,the modification promoting heterodimerization is in the heavy chain(s),specifically in the Fc domain, of an immunoglobulin molecule. In someembodiments the modification promoting heterodimerization comprises anamino acid mutation, specifically an amino acid substitution. In aparticular embodiment, the modification promoting heterodimerizationcomprises a separate amino acid mutation, specifically an amino acidsubstitution, in each of the two immunoglobulin heavy chains.

Similarly, a “modification promoting the association of the first andthe second subunit of the Fc domain” is a manipulation of the peptidebackbone or the post-translational modifications of an Fc domain subunitthat reduces or prevents the association of a polypeptide comprising theFc domain subunit with an identical polypeptide to form a homodimer. Amodification promoting association as used herein particularly includesseparate modifications made to each of the two Fc domain subunitsdesired to associate (i.e. the first and the second subunit of the Fcdomain), wherein the modifications are complementary to each other so asto promote association of the two Fc domain subunits. For example, amodification promoting association may alter the structure or charge ofone or both of the Fc domain subunits so as to make their associationsterically or electrostatically favorable, respectively. Thus,(hetero)dimerization occurs between a polypeptide comprising the firstFc domain subunit and a polypeptide comprising the second Fc domainsubunit, which might be non-identical in the sense that furthercomponents fused to each of the subunits (e.g. antigen binding moieties)are not the same. In some embodiments the modification promotingassociation comprises an amino acid mutation in the Fc domain,specifically an amino acid substitution. In a particular embodiment, themodification promoting association comprises a separate amino acidmutation, specifically an amino acid substitution, in each of the twosubunits of the Fc domain.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc region of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Activating Fcreceptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), andFcαRI (CD89).

The term “effector functions” when used in reference to antibodies referto those biological activities attributable to the Fc region of anantibody, which vary with the antibody isotype. Examples of antibodyeffector functions include: C1q binding and complement dependentcytotoxicity (CDC), Fc receptor binding, antibody-dependentcell-mediated cytotoxicity (ADCC), antibody-dependent cellularphagocytosis (ADCP), cytokine secretion, immune complex-mediated antigenuptake by antigen presenting cells, down regulation of cell surfacereceptors (e.g. B cell receptor), and B cell activation.

As used herein, the term “effector cells” refers to a population oflymphocytes that display effector moiety receptors, e.g. cytokinereceptors, and/or Fc receptors on their surface through which they bindan effector moiety, e.g. a cytokine, and/or an Fc region of an antibodyand contribute to the destruction of target cells, e.g. tumor cells.Effector cells may for example mediate cytotoxic or phagocytic effects.Effector cells include, but are not limited to, effector T cells such asCD8⁺ cytotoxic T cells, CD4⁺ helper T cells, γδ T cells, NK cells,lymphokine-activated killer (LAK) cells and macrophages/monocytes.

As used herein, the terms “engineer, engineered, engineering,” areconsidered to include any manipulation of the peptide backbone or thepost-translational modifications of a naturally occurring or recombinantpolypeptide or fragment thereof. Engineering includes modifications ofthe amino acid sequence, of the glycosylation pattern, or of the sidechain group of individual amino acids, as well as combinations of theseapproaches. “Engineering”, particularly with the prefix “glyco-”, aswell as the term “glycosylation engineering” includes metabolicengineering of the glycosylation machinery of a cell, including geneticmanipulations of the oligosaccharide synthesis pathways to achievealtered glycosylation of glycoproteins expressed in cells. Furthermore,glycosylation engineering includes the effects of mutations and cellenvironment on glycosylation. In one embodiment, the glycosylationengineering is an alteration in glycosyltransferase activity. In aparticular embodiment, the engineering results in alteredglucosaminyltransferase activity and/or fucosyltransferase activity.Glycosylation engineering can be used to obtain a “host cell havingincreased GnTIII activity” (e.g. a host cell that has been manipulatedto express increased levels of one or more polypeptides havingβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity), a “hostcell having increased ManII activity” (e.g. a host cell that has beenmanipulated to express increased levels of one or more polypeptideshaving α-mannosidase II (ManII) activity), or a “host cell havingdecreased α(1,6) fucosyltransferase activity” (e.g. a host cell that hasbeen manipulated to express decreased levels of α(1,6)fucosyltransferase).

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.A host cell is any type of cellular system that can be used to generateproteins used for the present invention. In one embodiment, the hostcell is engineered to allow the production of an antibody with modifiedoligosaccharides. In certain embodiments, the host cells have beenmanipulated to express increased levels of one or more polypeptideshaving β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity. Incertain embodiments the host cells have been further manipulated toexpress increased levels of one or more polypeptides havingα-mannosidase II (ManII) activity. Host cells include cultured cells,e.g. mammalian cultured cells, such as CHO cells, BHK cells, NS0 cells,SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells,PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plantcells, to name only a few, but also cells comprised within a transgenicanimal, transgenic plant or cultured plant or animal tissue.

As used herein, the term “polypeptide having GnTIII activity” refers topolypeptides that are able to catalyze the addition of aN-acetylglucosamine (GlcNAc) residue in β-1,4 linkage to the β-linkedmannoside of the thiomannosyl core of N-linked oligosaccharides. Thisincludes fusion polypeptides exhibiting enzymatic activity similar to,but not necessarily identical to, an activity ofβ(1,4)-N-acetylglucosaminyltransferase III, also known asβ-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyl-transferase (EC2.4.1.144), according to the Nomenclature Committee of the InternationalUnion of Biochemistry and Molecular Biology (NC-IUBMB), as measured in aparticular biological assay, with or without dose dependency. In thecase where dose dependency does exist, it need not be identical to thatof GnTIII, but rather substantially similar to the dose-dependency in agiven activity as compared to the GnTIII (i.e. the candidate polypeptidewill exhibit greater activity or not more than about 25-fold less and,preferably, not more than about ten-fold less activity, and mostpreferably, not more than about three-fold less activity relative to theGnTIII). In certain embodiments the polypeptide having GnTIII activityis a fusion polypeptide comprising the catalytic domain of GnTIII andthe Golgi localization domain of a heterologous Golgi residentpolypeptide. Particularly, the Golgi localization domain is thelocalization domain of mannosidase II or GnTI, most particularly thelocalization domain of mannosidase II. Alternatively, the Golgilocalization domain is selected from the group consisting of: thelocalization domain of mannosidase I, the localization domain of GnTII,and the localization domain of α1,6 core fucosyltransferase. Methods forgenerating such fusion polypeptides and using them to produce antibodieswith increased effector functions are disclosed in WO2004/065540, U.S.Provisional Pat. Appl. No. 60/495,142 and U.S. Pat. Appl. Publ. No.2004/0241817, the entire contents of which are expressly incorporatedherein by reference.

As used herein, the term “Golgi localization domain” refers to the aminoacid sequence of a Golgi resident polypeptide which is responsible foranchoring the polypeptide to a location within the Golgi complex.Generally, localization domains comprise amino terminal “tails” of anenzyme.

As used herein, the term “polypeptide having ManII activity” refers topolypeptides that are able to catalyze the hydrolysis of the terminal1,3- and 1,6-linked α-D-mannose residues in the branchedGlcNAcMan₅GlcNAc₂ mannose intermediate of N-linked oligosaccharides.This includes polypeptides exhibiting enzymatic activity similar to, butnot necessarily identical to, an activity of Golgi α-mannosidase II,also known as mannosyl oligosaccharide 1,3-1,6-α-mannosidase II (EC3.2.1.114), according to the Nomenclature Committee of the InternationalUnion of Biochemistry and Molecular Biology (NC-IUBMB).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immunemechanism leading to the lysis of antibody-coated target cells by immuneeffector cells. The target cells are cells to which antibodies orfragments thereof comprising an Fc region specifically bind, generallyvia the protein part that is N-terminal to the Fc region. As usedherein, the term “increased/reduced ADCC” is defined as either anincrease/reduction in the number of target cells that are lysed in agiven time, at a given concentration of antibody in the mediumsurrounding the target cells, by the mechanism of ADCC defined above,and/or a reduction/increase in the concentration of antibody, in themedium surrounding the target cells, required to achieve the lysis of agiven number of target cells in a given time, by the mechanism of ADCC.The increase/reduction in ADCC is relative to the ADCC mediated by thesame antibody produced by the same type of host cells, using the samestandard production, purification, formulation and storage methods(which are known to those skilled in the art), but that has not beenengineered. For example the increase in ADCC mediated by an antibodyproduced by host cells engineered to have an altered pattern ofglycosylation (e.g. to express the glycosyltransferase, GnTIII, or otherglycosyltransferases) by the methods described herein, is relative tothe ADCC mediated by the same antibody produced by the same type ofnon-engineered host cells.

By “antibody having increased/reduced antibody dependent cell-mediatedcytotoxicity (ADCC)” is meant an antibody having increased/reduced ADCCas determined by any suitable method known to those of ordinary skill inthe art. One accepted in vitro ADCC assay is as follows:

-   -   1) the assay uses target cells that are known to express the        target antigen recognized by the antigen-binding region of the        antibody;    -   2) the assay uses human peripheral blood mononuclear cells        (PBMCs), isolated from blood of a randomly chosen healthy donor,        as effector cells;    -   3) the assay is carried out according to following protocol:        -   i) the PBMCs are isolated using standard density            centrifugation procedures and are suspended at 5×10⁶            cells/ml in RPMI cell culture medium;        -   ii) the target cells are grown by standard tissue culture            methods, harvested from the exponential growth phase with a            viability higher than 90%, washed in RPMI cell culture            medium, labeled with 100 micro-Curies of ⁵¹Cr, washed twice            with cell culture medium, and resuspended in cell culture            medium at a density of 10⁵ cells/ml;        -   iii) 100 microliters of the final target cell suspension            above are transferred to each well of a 96-well microtiter            plate;        -   iv) the antibody is serially-diluted from 4000 ng/ml to 0.04            ng/ml in cell culture medium and 50 microliters of the            resulting antibody solutions are added to the target cells            in the 96-well microtiter plate, testing in triplicate            various antibody concentrations covering the whole            concentration range above;        -   v) for the maximum release (MR) controls, 3 additional wells            in the plate containing the labeled target cells, receive 50            microliters of a 2% (V/V) aqueous solution of non-ionic            detergent (Nonidet, Sigma, St. Louis), instead of the            antibody solution (point iv above);        -   vi) for the spontaneous release (SR) controls, 3 additional            wells in the plate containing the labeled target cells,            receive 50 microliters of RPMI cell culture medium instead            of the antibody solution (point iv above);        -   vii) the 96-well microtiter plate is then centrifuged at            50×g for 1 minute and incubated for 1 hour at 4° C.;        -   viii) 50 microliters of the PBMC suspension (point i above)            are added to each well to yield an effector:target cell            ratio of 25:1 and the plates are placed in an incubator            under 5% CO₂ atmosphere at 37° C. for 4 hours;        -   ix) the cell-free supernatant from each well is harvested            and the experimentally released radioactivity (ER) is            quantified using a gamma counter;        -   x) the percentage of specific lysis is calculated for each            antibody concentration according to the formula            (ER-MR)/(MR-SR)×100, where ER is the average radioactivity            quantified (see point ix above) for that antibody            concentration, MR is the average radioactivity quantified            (see point ix above) for the MR controls (see point v            above), and SR is the average radioactivity quantified (see            point ix above) for the SR controls (see point vi above);    -   4) “increased/reduced ADCC” is defined as either an        increase/reduction in the maximum percentage of specific lysis        observed within the antibody concentration range tested above,        and/or a reduction/increase in the concentration of antibody        required to achieve one half of the maximum percentage of        specific lysis observed within the antibody concentration range        tested above. The increase/reduction in ADCC is relative to the        ADCC, measured with the above assay, mediated by the same        antibody, produced by the same type of host cells, using the        same standard production, purification, formulation and storage        methods, which are known to those skilled in the art, but that        has not been engineered.

As used herein, the term “immunoconjugate” refers to a polypeptidemolecule that includes at least one effector moiety, such as a cytokine,and an antigen binding moiety, such as an antibody. In certainembodiments, the immunoconjugate comprises not more than one effectormoiety. Particular immunoconjugates useful in the invention essentiallyconsist of one effector moiety and an antibody joined by one or morepeptide linkers. Particular immunoconjugates according to the inventionare fusion proteins, i.e. the components of the immunoconjugate arejoined by peptide bonds.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

As used herein, the terms “first”, “second”, “third” etc. with respectto antigen binding moieties etc., are used for convenience ofdistinguishing when there is more than one of each type of moiety. Useof these terms is not intended to confer a specific order or orientationunless explicitly so stated.

The terms “multispecific” and “bispecific” mean that the antigen bindingmolecule is able to specifically bind to at least two distinct antigenicdeterminants. Typically, a bispecific antigen binding molecule comprisestwo antigen binding sites, each of which is specific for a differentantigenic determinant. In certain embodiments a bispecific antigenbinding molecule is capable of simultaneously binding two antigenicdeterminants, particularly two antigenic determinants expressed on twodistinct cells.

The term “valent” as used herein denotes the presence of a specifiednumber of antigen binding sites in an antigen binding molecule. As such,the term “monovalent binding to an antigen” denotes the presence of one(and not more than one) antigen binding site specific for the antigen inthe antigen binding molecule.

An “antigen binding site” refers to the site, i.e. one or more aminoacid residues, of an antigen binding molecule which provides interactionwith the antigen. For example, the antigen binding site of an antibodycomprises amino acid residues from the complementarity determiningregions (CDRs). A native immunoglobulin molecule typically has twoantigen binding sites, a Fab molecule typically has a single antigenbinding site.

A “T cell activating therapeutic agent” as used herein refers to atherapeutic agent capable of inducing T cell activation in a subject,particularly a therapeutic agent designed for inducing T-cell activationin a subject. Examples of T cell activating therapeutic agents includebispecific antibodies that specifically bind an activating T cellantigen, such as CD3, and a target cell antigen, such as CD20 or CD19.Further examples include chimeric antigen receptors (CARs) whichcomprise a T cell activating domain and an antigen binding moiety thatspecifically binds to a target cell antigen, such as CD20 or CD19.

An “activating T cell antigen” as used herein refers to an antigenicdeterminant expressed by a T lymphocyte, particularly a cytotoxic Tlymphocyte, which is capable of inducing or enhancing T cell activationupon interaction with an antigen binding molecule. Specifically,interaction of an antigen binding molecule with an activating T cellantigen may induce T cell activation by triggering the signaling cascadeof the T cell receptor complex. An exemplary activating T cell antigenis CD3.

“T cell activation” as used herein refers to one or more cellularresponse of a T lymphocyte, particularly a cytotoxic T lymphocyte,selected from: proliferation, differentiation, cytokine secretion,cytotoxic effector molecule release, cytotoxic activity, and expressionof activation markers. The T cell activating bispecific antigen bindingmolecules and T cell activating therapeutic agents used in the presentinvention are capable of inducing T cell activation. Suitable assays tomeasure T cell activation are known in the art described herein.

A “target cell antigen” as used herein refers to an antigenicdeterminant presented on the surface of a target cell, for example acell in a tumor such as a cancer cell or a cell of the tumor stroma.

A “B-cell antigen” as used herein refers to an antigenic determinantpresented on the surface of a B lymphocyte, particularly a malignant Blymphocyte (in that case the antigen also being referred to as“malignant B-cell antigen”).

A “T-cell antigen” as used herein refers to an antigenic determinantpresented on the surface of a T lymphocyte, particularly a cytotoxic Tlymphocyte.

A “Fab molecule” refers to a protein consisting of the VH and CH1 domainof the heavy chain (the “Fab heavy chain”) and the VL and CL domain ofthe light chain (the “Fab light chain”) of an immunoglobulin.

By “chimeric antigen receptor” or “CAR” is meant a geneticallyengineered receptor protein comprising an antigen binding moiety, e.g. asingle-chain variable fragment (scFv) of a targeting antibody, atransmembrane domain, an intracellular T-cell activating signalingdomain (e.g. the CD3 zeta chain of the T-cell receptor) and optionallyone or more intracellular co-stimulatory domains (e.g. of CD28, CD27,CD137 (4-1BB), Ox40). CARs mediate antigen recognition, T cellactivation, and—in the case of second-generation CARs—costimulation toaugment T cell functionality and persistence. For a review see e.g.Jackson et al., Nat Rev Clin Oncol (2016) 13, 370-383.

By “B cell proliferative disorder” is meant a disease wherein the numberof B cells in a patient is increased as compared to the number of Bcells in a healthy subject, and particularly wherein the increase in thenumber of B cells is the cause or hallmark of the disease. A“CD20-positive B cell proliferative disorder” is a B cell proliferativedisorder wherein B-cells, particularly malignant B-cells (in addition tonormal B-cells), express CD20. Exemplary B cell proliferation disordersinclude Non-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL),chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma(DLBCL), follicular lymphoma (FL), mantle-cell lymphoma (MCL), marginalzone lymphoma (MZL), as well as some types of Multiple myeloma (MM) andHodgkin lymphoma (HL).

By “fused” is meant that the components (e.g. a Fab molecule and an Fcdomain subunit) are linked by peptide bonds, either directly or via oneor more peptide linkers.

An “anti-drug antibody” or “ADA” refers to an antibody that binds to atherapeutic agent and may influence serum concentrations and function ofthe therapeutic agent in a subject. The presence of ADAs may increaseclearance of the therapeutic agent through formation of immune complexesbetween therapeutic agent and antibody (neutralizing, non-neutralizingor both), thus reducing the therapeutic agent's half-life. Furthermore,the activity and effectiveness of the therapeutic agent may be decreasedthrough binding of antibody to the therapeutic agent (particularly inthe case of neutralizing ADAs). ADAs can also be associated withallergic or hypersensitivity reactions and other adverse events.

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

By “therapeutic agent” is meant an active ingredient, e.g. of apharmaceutical composition, that is administered to a subject in anattempt to alter the natural course of a disease in the subject beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. An “immunotherapeutic agent” refers to atherapeutic agent that is administered to a subject in an attempt torestore or enhance the subject's immune response, e.g. to a tumor.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Preferably, the individual orsubject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe composition would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of a disease in the individual being treated,and can be performed either for prophylaxis or during the course ofclinical pathology. Desirable effects of treatment include, but are notlimited to, preventing occurrence or recurrence of disease, alleviationof symptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, methods of theinvention are used to delay development of a disease or to slow theprogression of a disease.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“CD3” refers to any native CD3 from any vertebrate source, includingmammals such as primates (e.g. humans), non-human primates (e.g.cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwiseindicated. The term encompasses “full-length,” unprocessed CD3 as wellas any form of CD3 that results from processing in the cell. The termalso encompasses naturally occurring variants of CD3, e.g., splicevariants or allelic variants. In one embodiment, CD3 is human CD3,particularly the epsilon subunit of human CD3 (CD3ε). The amino acidsequence of human CD3ε is shown in UniProt (www.uniprot.org) accessionno. P07766 (version 144), or NCBI (www.ncbi.nlm.nih.gov/) RefSeqNP_000724.1. See also SEQ ID NO: 115. The amino acid sequence ofcynomolgus [Macaca fascicularis] CD3ε is shown in NCBI GenBank no.BAB71849.1. See also SEQ ID NO: 116.

“CD19” refers to B-lymphocyte antigen CD19, also known as B-lymphocytesurface antigen B4 or T-cell surface antigen Leu-12 and includes anynative CD19 from any vertebrate source, including mammals such asprimates (e.g. humans) and rodents (e.g., mice and rats), unlessotherwise indicated. The term encompasses “full-length,” unprocessedCD19 as well as any form of CD19 that results from processing in thecell. The term also encompasses naturally occurring variants of CD19,e.g., splice variants or allelic variants. In one embodiment, CD19 ishuman CD19. The amino acid sequence of an exemplary human CD19 is shownin UniProt (www.uniprot.org) accession no. P15391 (version 174), or NCBI(www.ncbi.nlm.nih.gov/) RefSeq NP_001770.5, and SEQ ID NO: 117.

“Carcinoembryonic antigen” or “CEA” (also known as Carcinoembryonicantigen-related cell adhesion molecule 5 (CEACAM5)) refers to any nativeCEA from any vertebrate source, including mammals such as primates (e.g.humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g.mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed CEA as well as any form of CEA that resultsfrom processing in the cell. The term also encompasses naturallyoccurring variants of CEA, e.g., splice variants or allelic variants. Inone embodiment, CEA is human CEA. The amino acid sequence of human CEAis shown in UniProt (www.uniprot.org) accession no. P06731, or NCBI(www.ncbi.nlm.nih.gov/) RefSeq NP_004354.2.

“Fibroblast activation protein” or “FAP” (also known as seprase) refersto any native FAP from any vertebrate source, including mammals such asprimates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) androdents (e.g. mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed FAP as well as any form of FAPthat results from processing in the cell. The term also encompassesnaturally occurring variants of FAP, e.g., splice variants or allelicvariants. In one embodiment, FAP is human FAP. The amino acid sequenceof human FAP is shown in UniProt (www.uniprot.org) accession no. Q12884,or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_004451.2.

By a “crossover” Fab molecule (also termed “Crossfab”) is meant a Fabmolecule wherein the variable domains or the constant domains of the Fabheavy and light chain are exchanged (i.e. replaced by each other), i.e.the crossover Fab molecule comprises a peptide chain composed of thelight chain variable domain VL and the heavy chain constant domain 1 CH1(VL-CH1, in N- to C-terminal direction), and a peptide chain composed ofthe heavy chain variable domain VH and the light chain constant domainCL (VH-CL, in N- to C-terminal direction). For clarity, in a crossoverFab molecule wherein the variable domains of the Fab light chain and theFab heavy chain are exchanged, the peptide chain comprising the heavychain constant domain 1 CH1 is referred to herein as the “heavy chain”of the (crossover) Fab molecule. Conversely, in a crossover Fab moleculewherein the constant domains of the Fab light chain and the Fab heavychain are exchanged, the peptide chain comprising the heavy chainvariable domain VH is referred to herein as the “heavy chain” of the(crossover) Fab molecule.

In contrast thereto, by a “conventional” Fab molecule is meant a Fabmolecule in its natural format, i.e. comprising a heavy chain composedof the heavy chain variable and constant domains (VH-CH1, in N- toC-terminal direction), and a light chain composed of the light chainvariable and constant domains (VL-CL, in N- to C-terminal direction).

Type II Anti-CD20 Antibodies

The CD20 molecule (also called human B-lymphocyte-restricteddifferentiation antigen or Bp35) is a hydrophobic transmembrane proteinexpressed on the surface of malignant and non-malignant pre-B and matureB lymphocytes that has been described extensively (Valentine, M. A., etal., J. Biol. Chem. 264 (1989) 11282-11287; and Einfeld, D. A., et al.,EMBO J. 7 (1988) 711-717; Tedder, T. F., et al., Proc. Natl. Acad. Sci.U.S.A. 85 (1988) 208-212; Stamenkovic, I., et al., J. Exp. Med. 167(1988) 1975-1980; Tedder, T. F., et al., J. Immunol. 142 (1989)2560-2568).

CD20 is highly expressed by over 90% of B cell non-Hodgkin's lymphomas(NHL) (Anderson, K. C., et al., Blood 63 (1984) 1424-1433) but is notfound on hematopoietic stem cells, pro-B cells, normal plasma cells, orother normal tissues (Tedder, T. F., et al., J, Immunol. 135 (1985)973-979).

There exist two different types of anti-CD20 antibodies differingsignificantly in their mode of CD20 binding and biological activities(Cragg, M. S., et al., Blood 103 (2004) 2738-2743; and Cragg, M. S., etal., Blood 101 (2003) 1045-1052). Type I anti-CD20 antibodies primarilyutilize complement to kill target cells, while Type II antibodiesprimarily operate through direct induction of cell death.

Type I and Type II anti-CD20 antibodies and their characteristics arereviewed e.g. in Klein et al., mAbs 5 (2013), 22-33. Type II anti-CD20antibodies do not localize CD20 to lipid rafts, show low CDC activity,show only about half the binding capacity to B cells as compared to TypeI anti-CD20 antibodies, and induce homotypic aggregation and direct celldeath. In contrast thereto, Type I antibodies localize CD20 to lipidrafts, show high CDC activity, full binding capacity to B cells, andonly weak induction of homotypic aggregation and direct cell death.

Obinutuzumab and tositumomab (CAS number 192391-48) are examples of TypeII anti-CD20 antibodies, while rituximab, ofatumumab, veltuzumab,ocaratuzumab, ocrelizumab, PRO131921 and ublituximab are examples ofType I anti-CD20 antibodies.

According to the invention, the anti-CD20 antibody is a Type IIanti-CD20 antibody. In one embodiment according to the presentinvention, the Type II anti-CD20 antibody is capable of reducing thenumber of B cells in a subject. In one embodiment the Type II anti-CD20antibody is an IgG antibody, particularly an IgG1 antibody. In oneembodiment, the Type II anti-CD20 antibody is a full-length antibody. Inone embodiment, the Type II anti-CD20 antibody comprises an Fc region,particularly an IgG Fc region or, more particularly, an IgG1 Fc region.In one embodiment the Type II anti-CD20 antibody is a humanized B-Ly1antibody. Particularly, the Type II anti-CD20 antibody is a humanized,IgG-class Type II anti-CD20 antibody, having the binding specificity ofthe murine B-Ly1 antibody (Poppema and Visser, Biotest Bulletin 3,131-139 (1987); SEQ ID NOs 2 and 3).

In one embodiment, the Type II anti-CD20 antibody comprises a heavychain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ IDNO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and alight chain variable region comprising the light chain CDR (LCDR) 1 ofSEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.Particularly, the heavy chain variable region framework regions (FRs)FR1, FR2, and FR3 of said Type II anti-CD20 antibody are human FRsequences encoded by the VH1_10 human germ-line sequence, the heavychain variable region FR4 of said anti-CD20 antibody is a human FRsequence encoded by the JH4 human germ-line sequence, the light chainvariable region FRs FR1, FR2, and FR3 of said Type II anti-CD20 antibodyare human FR sequences encoded by the VK_2_40 human germ-line sequence,and the light chain variable region FR4 of said anti-CD20 antibody is ahuman FR sequence encoded by the JK4 human germ-line sequence. In oneembodiment, the Type II anti-CD20 antibody comprises the heavy chainvariable region sequence of SEQ ID NO: 10 and the light chain variableregion sequence of SEQ ID NO: 11.

In a particular embodiment, the Type II anti-CD20 antibody isobinutuzumab (recommended INN, WHO Drug Information, Vol. 26, No. 4,2012, p. 453). As used herein, obinutuzumab is synonymous for GA101. Thetradename is GAZYVA® or GAZYVARO®. This replaces all previous versions(e.g. Vol. 25, No. 1, 2011, p. 75-76), and is formerly known asafutuzumab (recommended INN, WHO Drug Information, Vol. 23, No. 2, 2009,p. 176; Vol. 22, No. 2, 2008, p. 124). In one embodiment, the Type IIanti-CD20 antibody is tositumomab.

The Type II anti-CD20 antibody useful in the present invention may beengineered to have increased effector function, as compared to acorresponding non-engineered antibody. In one embodiment the antibodyengineered to have increased effector function has at least 2-fold, atleast 10-fold or even at least 100-fold increased effector function,compared to a corresponding non-engineered antibody. The increasedeffector function can include, but is not limited to, one or more of thefollowing: increased Fc receptor binding, increased C1q binding andcomplement dependent cytotoxicity (CDC), increased antibody-dependentcell-mediated cytotoxicity (ADCC), increased antibody-dependent cellularphagocytosis (ADCP), increased cytokine secretion, increased immunecomplex-mediated antigen uptake by antigen-presenting cells, increasedbinding to NK cells, increased binding to macrophages, increased bindingto monocytes, increased binding to polymorphonuclear cells, increaseddirect signaling inducing apoptosis, increased crosslinking oftarget-bound antibodies, increased dendritic cell maturation, orincreased T cell priming.

In one embodiment the increased effector function one or more selectedfrom the group of increased Fc receptor binding, increased CDC,increased ADCC, increased ADCP, and increased cytokine secretion. In oneembodiment the increased effector function is increased binding to anactivating Fc receptor. In one such embodiment the binding affinity tothe activating Fc receptor is increased at least 2-fold, particularly atleast 10-fold, compared to the binding affinity of a correspondingnon-engineered antibody. In a specific embodiment the activating Fcreceptor is selected from the group of FcγRIIIa, FcγRI, and FcγRIIa. Inone embodiment the activating Fc receptor is FcγRIIIa, particularlyhuman FcγRIIIa. In another embodiment the increased effector function isincreased ADCC. In one such embodiment the ADCC is increased at least10-fold, particularly at least 100-fold, compared to the ADCC mediatedby a corresponding non-engineered antibody. In yet another embodimentthe increased effector function is increased binding to an activating Fcreceptor and increased ADCC.

Increased effector function can be measured by methods known in the art.A suitable assay for measuring ADCC is described herein. Other examplesof in vitro assays to assess ADCC activity of a molecule of interest aredescribed in U.S. Pat. No. 5,500,362; Hellstrom et al. Proc Natl AcadSci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad SciUSA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., JExp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assaysmethods may be employed (see, for example, ACTI™ non-radioactivecytotoxicity assay for flow cytometry (CellTechnology, Inc. MountainView, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega,Madison, WI)). Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g. in a animal model such as thatdisclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. According to a particularembodiment, binding affinity to an activating Fc receptor is measured bysurface plasmon resonance using a BIACORE® T100 machine (GE Healthcare)at 25° C. Alternatively, binding affinity of antibodies for Fc receptorsmay be evaluated using cell lines known to express particular Fcreceptors, such as NK cells expressing FcγIIIa receptor. C1q bindingassays may also be carried out to determine whether the antibody is ableto bind C1q and hence has CDC activity. See e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al.,Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743(2004)).

Increased effector function may result e.g. from glycoengineering of theFc region or the introduction of amino acid mutations in the Fc regionof the antibody. In one embodiment the anti-CD20 antibody is engineeredby introduction of one or more amino acid mutations in the Fc region. Ina specific embodiment the amino acid mutations are amino acidsubstitutions. In an even more specific embodiment the amino acidsubstitutions are at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues). Further suitable amino acid mutations aredescribed e.g. in Shields et al., J Biol Chem 9(2), 6591-6604 (2001);U.S. Pat. No. 6,737,056; WO 2004/063351 and WO 2004/099249. Mutant Fcregions can be prepared by amino acid deletion, substitution, insertionor modification using genetic or chemical methods well known in the art.Genetic methods may include site-specific mutagenesis of the encodingDNA sequence, PCR, gene synthesis, and the like. The correct nucleotidechanges can be verified for example by sequencing.

In another embodiment the Type II anti-CD20 antibody is engineered bymodification of the glycosylation in the Fc region. In a specificembodiment the Type II anti-CD20 antibody is engineered to have anincreased proportion of non-fucosylated oligosaccharides in the Fcregion as compared to a non-engineered antibody. An increased proportionof non-fucosylated oligosaccharides in the Fc region of an antibodyresults in the antibody having increased effector function, inparticular increased ADCC.

In a more specific embodiment, at least about 20%, about 25%, about 30%,about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,or about 100%, preferably at least about 40%, of the N-linkedoligosaccharides in the Fc region of the Type II anti-CD20 antibody arenon-fucosylated. In one embodiment, between about 40% and about 80% ofthe N-linked oligosaccharides in the Fc region of the Type II anti-CD20antibody are non-fucosylated. In one embodiment, between about 40% andabout 60% of the N-linked oligosaccharides in the Fc region of the TypeII anti-CD20 antibody are non-fucosylated. The non-fucosylatedoligosaccharides may be of the hybrid or complex type.

In another specific embodiment the Type II anti-CD20 antibody isengineered to have an increased proportion of bisected oligosaccharidesin the Fc region as compared to a non-engineered antibody. In a morespecific embodiment, at least about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, or about 100%, preferably at least about 40%, of theN-linked oligosaccharides in the Fc region of the Type II anti-CD20antibody are bisected. In one embodiment, between about 40% and about80% of the N-linked oligosaccharides in the Fc region of the anti-CD20antibody are bisected. In one embodiment, between about 40% and about60% of the N-linked oligosaccharides in the Fc region of the Type IIanti-CD20 antibody are bisected. The bisected oligosaccharides may be ofthe hybrid or complex type.

In yet another specific embodiment the anti-CD20 antibody is engineeredto have an increased proportion of bisected, non-fucosylatedoligosaccharides in the Fc region, as compared to a non-engineeredantibody. In a more specific embodiment, at least about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, or about 100%, preferably at leastabout 15%, more preferably at least about 25%, of the N-linkedoligosaccharides in the Fc region of the anti-CD20 antibody arebisected, non-fucosylated. The bisected, non-fucosylatedoligosaccharides may be of the hybrid or complex type.

The oligosaccharide structures in the antibody Fc region can be analysedby methods well known in the art, e.g. by MALDI TOF mass spectrometry asdescribed in Umana et al., Nat Biotechnol 17, 176-180 (1999) or Ferraraet al., Biotechn Bioeng 93, 851-861 (2006). The percentage ofnon-fucosylated oligosaccharides is the amount of oligosaccharideslacking fucose residues, relative to all oligosaccharides attached toAsn 297 (e. g. complex, hybrid and high mannose structures) andidentified in an N-glycosidase F treated sample by MALDI TOF MS. Asn 297refers to the asparagine residue located at about position 297 in the Fcregion (EU numbering of Fc region residues); however, Asn297 may also belocated about ±3 amino acids upstream or downstream of position 297,i.e., between positions 294 and 300, due to minor sequence variations inantibodies. The percentage of bisected, or bisected non-fucosylated,oligosaccharides is determined analogously.

In one embodiment the Type II anti-CD20 antibody is engineered to havemodified glycosylation in the Fc region, as compared to a non-engineeredantibody, by producing the antibody in a host cell having alteredactivity of one or more glycosyltransferase. Glycosyltransferasesinclude β(1,4)-N-acetylglucosaminyltransferase III (GnTIII),β(1,4)-galactosyltransferase (GalT),β(1,2)-N-acetylglucosaminyltransferase I (GnTI),β(1,2)-N-acetylglucosaminyltransferase II (GnTII) andα(1,6)-fucosyltransferase. In a specific embodiment the Type IIanti-CD20 antibody is engineered to have an increased proportion ofnon-fucosylated oligosaccharides in the Fc region, as compared to anon-engineered antibody, by producing the antibody in a host cell havingincreased β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity.In an even more specific embodiment the host cell additionally hasincreased α-mannosidase II (ManII) activity. The glycoengineeringmethodology that can be used for engineering antibodies useful for thepresent invention has been described in greater detail in Umana et al.,Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93,851-861 (2006); WO 99/54342 (U.S. Pat. No. 6,602,684; EP 1071700); WO2004/065540 (U.S. Pat. Appl. Publ. No. 2004/0241817; EP 1587921), WO03/011878 (U.S. Pat. Appl. Publ. No. 2003/0175884), the entire contentof each of which is incorporated herein by reference in its entirety.Antibodies glycoengineered using this methodology are referred to asGlycoMabs herein.

Generally, any type of cultured cell line, including the cell linesdiscussed herein, can be used to generate cell lines for the productionof anti-TNC A2 antibodies with altered glycosylation pattern. Particularcell lines include CHO cells, BHK cells, NS0 cells, SP2/0 cells, YOmyeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells orhybridoma cells, and other mammalian cells. In certain embodiments, thehost cells have been manipulated to express increased levels of one ormore polypeptides having β(1,4)-N-acetylglucosaminyltransferase III(GnTIII) activity. In certain embodiments the host cells have beenfurther manipulated to express increased levels of one or morepolypeptides having α-mannosidase II (ManII) activity. In a specificembodiment, the polypeptide having GnTIII activity is a fusionpolypeptide comprising the catalytic domain of GnTIII and the Golgilocalization domain of a heterologous Golgi resident polypeptide.Particularly, said Golgi localization domain is the Golgi localizationdomain of mannosidase II. Methods for generating such fusionpolypeptides and using them to produce antibodies with increasedeffector functions are disclosed in Ferrara et al., Biotechn Bioeng 93,851-861 (2006) and WO2004/065540, the entire contents of which areexpressly incorporated herein by reference.

The host cells which contain the coding sequence of an antibody usefulfor the invention and/or the coding sequence of polypeptides havingglycosyltransferase activity, and which express the biologically activegene products may be identified e.g. by DNA-DNA or DNA-RNAhybridization; the presence or absence of “marker” gene functions;assessing the level of transcription as measured by the expression ofthe respective mRNA transcripts in the host cell; or detection of thegene product as measured by immunoassay or by its biologicalactivity—methods which are well known in the art. GnTIII or Man IIactivity can be detected e.g. by employing a lectin which binds tobiosynthesis products of GnTIII or ManII, respectively. An example forsuch a lectin is the E₄-PHA lectin which binds preferentially tooligosaccharides containing bisecting GlcNAc. Biosynthesis products(i.e. specific oligosaccharide structures) of polypeptides having GnTIIIor ManII activity can also be detected by mass spectrometric analysis ofoligosaccharides released from glycoproteins produced by cellsexpressing said polypeptides. Alternatively, a functional assay whichmeasures the increased effector function, e.g. increased Fc receptorbinding, mediated by antibodies produced by the cells engineered withthe polypeptide having GnTIII or ManII activity may be used.

In another embodiment the anti-CD20 antibody is engineered to have anincreased proportion of non-fucosylated oligosaccharides in the Fcregion, as compared to a non-engineered antibody, by producing theantibody in a host cell having decreased α(1,6)-fucosyltransferaseactivity. A host cell having decreased α(1,6)-fucosyltransferaseactivity may be a cell in which the α(1,6)-fucosyltransferase gene hasbeen disrupted or otherwise deactivated, e.g. knocked out (seeYamane-Ohnuki et al., Biotech Bioeng 87, 614 (2004); Kanda et al.,Biotechnol Bioeng, 94(4), 680-688 (2006); Niwa et al., J Immunol Methods306, 151-160 (2006)).

Other examples of cell lines capable of producing defucosylatedantibodies include Lec13 CHO cells deficient in protein fucosylation(Ripka et al., Arch Biochem Biophys 249, 533-545 (1986); US Pat. Appl.No. US 2003/0157108; and WO 2004/056312, especially at Example 11). Theantibodies useful in the present invention can alternatively beglycoengineered to have reduced fucose residues in the Fc regionaccording to the techniques disclosed in EP 1 176 195 A1, WO 03/084570,WO 03/085119 and U.S. Pat. Appl. Pub. Nos. 2003/0115614, 2004/093621,2004/110282, 2004/110704, 2004/132140, U.S. Pat. No. 6,946,292 (Kyowa),e.g. by reducing or abolishing the activity of a GDP-fucose transporterprotein in the host cells used for antibody production.

Glycoengineered antibodies useful in the invention may also be producedin expression systems that produce modified glycoproteins, such as thosetaught in WO 03/056914 (GlycoFi, Inc.) or in WO 2004/057002 and WO2004/024927 (Greenovation).

Therapeutic Agents

The present invention is useful in connection with various therapeuticagents, particularly with therapeutic agents that are immunogenic in thesubject (i.e. have the ability of inducing an immune response in thesubject) and/or that activate T-cells in the subject. Such therapeuticagents include, for example, recombinant proteins.

In one embodiment, the therapeutic agent induces the formation of ADAsin a subject when administered to the subject in a treatment regimenwithout the administration of a Type II anti-CD20 antibody. In oneembodiment, the therapeutic agent induces cytokine release in a subjectwhen administered to the subject in a treatment regimen without theadministration of a Type II anti-CD20 antibody. In one embodiment, thetherapeutic agent induces formation of ADAs and cytokine release in asubject when administered to the subject in a treatment regimen withoutthe administration of a Type II anti-CD20 antibody.

In one embodiment, the therapeutic agent is a biologic agent. In oneembodiment, the therapeutic agent comprises a polypeptide, particularlya recombinant polypeptide. In one embodiment, the therapeutic agentcomprises a polypeptide that does not naturally occur in the subjectand/or is immunogenic in the subject. In one embodiment, the therapeuticagent is to be systemically administered. In one embodiment, thetherapeutic agent is to be administered by infusion, particularlyintravenous infusion.

In one embodiment, the therapeutic agent comprises an antigen bindingpolypeptide. In one embodiment, the therapeutic agent comprises apolypeptide selected from the group of an antibody, an antibodyfragment, an Fc domain, and an immunoconjugate. In one embodiment, thetherapeutic agent comprises a polypeptide selected from the group of anantibody, an antibody fragment, an antigen receptor or anantigen-binding fragment thereof, and a receptor ligand or areceptor-binding fragment thereof. In one embodiment, the therapeuticagent comprises an antibody. In one embodiment, the antibody is amonoclonal antibody. In one embodiment, the antibody is a polyclonalantibody. In one embodiment the antibody is a human antibody. In oneembodiment, the antibody is humanized antibody. In one embodiment theantibody is a chimeric antibody. In one embodiment the antibody isfull-length antibody. In one embodiment the antibody is an IgG-classantibody, particularly an IgG1 subclass antibody. In one embodiment, theantibody is a recombinant antibody.

In certain embodiments, the therapeutic agent comprises an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Plückthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046. In one embodiment, the antibodyfragment is a Fab fragment or a scFv fragment.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

In certain embodiments, the therapeutic agent comprises a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, the therapeutic agent comprises a humanizedantibody. Typically, a non-human antibody is humanized to reduceimmunogenicity to humans, while retaining the specificity and affinityof the parental non-human antibody. Generally, a humanized antibodycomprises one or more variable domains in which HVRs, e.g., CDRs, (orportions thereof) are derived from a non-human antibody, and FRs (orportions thereof) are derived from human antibody sequences. A humanizedantibody optionally will also comprise at least a portion of a humanconstant region. In some embodiments, some FR residues in a humanizedantibody are substituted with corresponding residues from a non-humanantibody (e.g., the antibody from which the HVR residues are derived),e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing specificity determining region(SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimkaet al., Br. J. Cancer, 83:252-260 (2000) (describing the “guidedselection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

In certain embodiments, the therapeutic agent comprises a humanantibody. Human antibodies can be produced using various techniquesknown in the art. Human antibodies are described generally in van Dijkand van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg,Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HuMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELocIMousE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al. Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).Additional methods include those described, for example, in U.S. Pat.No. 7,189,826 (describing production of monoclonal human IgM antibodiesfrom hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268(2006) (describing human-human hybridomas). Human hybridoma technology(Trioma technology) is also described in Vollmers and Brandlein,Histology and Histopathology, 20(3):927-937 (2005) and Vollmers andBrandlein, Methods and Findings in Experimental and ClinicalPharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

Antibodies comprised in the therapeutic agent may be isolated byscreening combinatorial libraries for antibodies with the desiredactivity or activities. For example, a variety of methods are known inthe art for generating phage display libraries and screening suchlibraries for antibodies possessing the desired binding characteristics.Such methods are reviewed, e.g., in Hoogenboom et al. in Methods inMolecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa,NJ, 2001) and further described, e.g., in the McCafferty et al., Nature348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al.,J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods inMolecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003);Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol.Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self-antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

In certain embodiments, the therapeutic agent comprises a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, the binding specificities arefor different antigens. In certain embodiments, the bindingspecificities are for different epitopes on the same antigen. Bispecificantibodies may also be used to localize cytotoxic agents to cells whichexpress an antigen. Bispecific antibodies can be prepared as full lengthantibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to two differentantigens (see, US 2008/0069820, for example).

“Crossmab” antibodies are also included herein (see e.g. WO2009080251,WO2009080252, WO2009080253, WO2009080254).

Another technique for making bispecific antibody fragments is the“bispecific T cell engager” or BiTE® approach (see, e.g., WO2004/106381,WO2005/061547, WO2007/042261 and WO2008/119567). This approach utilizestwo antibody variable domains arranged on a single polypeptide. Forexample, a single polypeptide chain includes two single chain Fv (scFv)fragments, each having a variable heavy chain (VH) and a variable lightchain (VL) domain separated by a polypeptide linker of a lengthsufficient to allow intramolecular association between the two domains.This single polypeptide further includes a polypeptide spacer sequencebetween the two scFv fragments, Each scFv recognizes a differentepitope, and these epitopes may be specific for different cell types,such that cells of two different cell types are brought into closeproximity or tethered when each scFv is engaged with its cognateepitope. One particular embodiment of this approach includes a ssFvrecognizing a cell-surface antigen expressed by an immune cell, e.g., aCD3 polypeptide on a T cell, linked to another scFv that recognizes acell-surface antigen expressed by a target cell, such as a malignant ortumor cell.

As it is a single polypeptide, the bispecific T cell engager may beexpressed using any prokaryotic or eukaryotic cell expression systemknown in the art, e.g., a CHO cell line. However, specific purificationtechniques (see, e.g., EP1691833) may be necessary to separate monomericbispecific T cell engagers from other multimeric species, which may havebiological activities other than the intended activity of the monomer.In one exemplary purification scheme, a solution containing secretedpolypeptides is first subjected to a metal affinity chromatography, andpolypeptides are eluted with a gradient of imidazole concentrations.This eluate is further purified using anion exchange chromatography, andpolypeptides are eluted using with a gradient of sodium chlorideconcentrations. Finally, this eluate is subjected to size exclusionchromatography to separate monomers from multimeric species.

Antibodies with more than t valencies are contemplated. For example,trispecific antibodies can be prepared. Tuft et al. J. Immunol. 147: 60(1991).

In certain embodiments, an antibody comprised in the therapeutic agentmay be further modified to contain additional nonproteinaceous moietiesthat are known in the art and readily available. The moieties suitablefor derivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethyleneglycol propionaldehyde may have advantages in manufacturing due to itsstability in water. The polymer may be of any molecular weight, and maybe branched or unbranched. The number of polymers attached to theantibody may vary, and if more than one polymer are attached, they canbe the same or different molecules. In general, the number and/or typeof polymers used for derivatization can be determined based onconsiderations including, but not limited to, the particular propertiesor functions of the antibody to be improved, whether the antibodyderivative will be used in a therapy under defined conditions, etc.

The therapeutic agent may also comprise an antibody conjugated to one ormore cytotoxic agents, such as chemotherapeutic agents or drugs, growthinhibitory agents, toxins (e.g., protein toxins, enzymatically activetoxins of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or radioactive isotopes.

In one embodiment, the therapeutic agent comprises an antibody-drugconjugate (ADC) in which an antibody is conjugated to one or more drugs,including but not limited to a maytansinoid (see U.S. Pat. Nos.5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an auristatinsuch as monomethylauristatin drug moieties DE and DF (MMAE and MMAF)(see U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); adolastatin; a calicheamicin or derivative thereof (see U.S. Pat. Nos.5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342(1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); ananthracycline such as daunomycin or doxorubicin (see Kratz et al.,Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med.Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem.16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834(2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532(2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat.No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel,paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.

In another embodiment, the therapeutic agent comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, the therapeutic agent comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example Tc^(99m) orI¹²³, or a spin label for nuclear magnetic resonance (NMR) imaging (alsoknown as magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

In some embodiments, the therapeutic agent may comprise an monoclonalantibody such as, but not limited to, alemtuzumab (LEMTRADA®),bevacizumab (AVASTIN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®),pertuzumab (OMNITARG®, 2C4), trastuzumab (HERCEPTIN®), tositumomab(Bexxar®), abciximab (REOPRO®), adalimumab (HUMIRA®), apolizumab,aselizumab, atlizumab, bapineuzumab, basiliximab (SIMULECT®),bavituximab, belimumab (BENLYSTA®) briakinumab, canakinumab (MARIS®),cedelizumab, certolizumab pegol (CIMZIA®), cidfusituzumab, cidtuzumab,cixutumumab, clazakizumab, crenezumab, daclizumab (ZENAPAX®),dalotuzumab, denosumab (PROLIA®, XGEVA®), eculizumab (SOLIRIS®),efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, golimumab(SIMPONI®), ipilimumab, imgatuzumab, infliximab (REMICADE®),labetuzumab, lebrikizumab, lexatumumab, lintuzumab, lucatumumab,lulizumab pegol, lumretuzumab, mapatumumab, matuzumab, mepolizumab,mogamulizumab, motavizumab, motovizumab, muromonab, natalizumab(TYSABRI®), necitumumab (PORTRAZZA®), nimotuzumab (THERACIM®),nolovizumab, numavizumab, olokizumab, ornalizumab (XOLAIR®), onartuzumab(also known as MetMAb), palivizumab (SYNAGIS®), pascolizumab,pecfusituzumab, pectuzumab, pembrolizumab (KEYTRUDA®), pexelizumab,priliximab, ralivizumab, ranibizumab (LUCENTIS®), reslivizumab,reslizumab, resyvizumab, robatumumab, rontalizumab, rovelizumab,ruplizumab, sarilumab, secukinumab, seribantumab, sifalimumab,sibrotuzumab, siltuximab (SYLVANT®) siplizumab, sontuzumab, tadocizumab,talizumab, tefibazumab, tocilizumab (ACTEMRA®), toralizuniab,tucusituzumab, umavizumab, urtoxazumab, ustekinumab (STELARA®),vedolizumab (ENTYVIO®), visilizumab, zanolimumab, zaluturnumab.

In one embodiment, the therapeutic agent comprises an antibody indicatedfor the treatment of cancer. In one embodiment, the therapeutic agentcomprises an antibody indicated for the treatment of an autoimmunedisease. In one embodiment, the therapeutic agent is animmunotherapeutic agent. In one embodiment the therapeutic agent isindicated for the treatment of cancer. In some embodiments, inparticular in relation aspects of the invention concerned with thereduction of cytokine release associated with the administration of atherapeutic agent in a subject, the cancer is a B-cell proliferativedisorder. In one embodiment, the cancer is a CD20-positive B-cellproliferative disorder. In one embodiment, the cancer is selected fromthe group consisting of Non-Hodgkin lymphoma (NHL), acute lymphocyticleukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-celllymphoma (DLBCL), follicular lymphoma (FL), mantle-cell lymphoma (MCL),marginal zone lymphoma (MZL), Multiple myeloma (MM), and Hodgkinlymphoma (HL). In one embodiment, the therapeutic agent is animmunotherapeutic agent.

In one embodiment, the therapeutic agent is an immunosuppressive agent.In one embodiment, the therapeutic agent is indicated for the treatmentof an autoimmune disease.

Without wishing to be bound theory, it is thought that enhancing T cellstimulation, by promoting an activating co-stimulatory molecule or byinhibiting a negative co-stimulatory molecule, may promote tumor celldeath thereby treating or delaying progression of cancer. In someembodiments, the therapeutic agent may comprise an agonist directedagainst an activating co-stimulatory molecule. In some embodiments, anactivating co-stimulatory molecule may include CD40, CD226, CD28, OX40,GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the agonistdirected against an activating co-stimulatory molecule is an agonistantibody that binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM,or CD127. In some embodiments, the therapeutic agent may comprise anantibody targeting GITR. In some embodiments, the antibody targetingGITR is TRX518. In some the therapeutic agent may comprise an antagonistdirected against an inhibitory co-stimulatory molecule. In someembodiments, an inhibitory co-stimulatory molecule may include CTLA-4(also known as CIA 52), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4,IDO, TIGIT, MICA/B, or arginase. In some embodiments, the antagonistdirected against an inhibitory co-stimulatory molecule is an antagonistantibody that binds to CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3,B7-H4, IDO, MICA/B, or arginase.

In some embodiments, the therapeutic agent may comprise an anti-PD-1antibody. In one embodiment the anti-PD-1 antibody is selected from thegroup consisting of MDX-1106 (nivolumab), MK-3475 (pembrolizumab,formerly known as lambrolizumab), CT-011 (pidilizumab). IDX-1106, alsoknown as MDX-1106-04, ONO-4538, BMS-936558, or nivolumab, is ananti-PD-1 antibody described in WO2006/121168. MK-3475, also known aspembrolizumab or (formerly) lambrolizumab, is an anti-PD-1 antibodydescribed in WO2009/114335. CT-011, also known as hBAT, hBAT-1 orpidilizumab, is an anti-PD-1 antibody described in WO2009/101 611.

In some embodiments, the therapeutic agent may comprise an immunoadhesinan immunoadhesin comprising an extracellular or PD-1 binding portion ofPD-L1 or PD-L2 fused to a constant region (e.g., an Fe region of animmunoglobulin sequence)). In one embodiment, the therapeutic agent maycomprise AMP-224, also known as B7-DCIg (a. PD-L2-Fc fusion solublereceptor described in WO2010/027827 and WO2011/066342).

In some embodiments, the therapeutic agent may comprise an anti-PD-L1antibody. In one embodiment the anti-PD-L1 antibody is selected from thegroup consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MED14736.Antibody YW243.55.S70 is an anti-PD-L1 antibody described in WO2010/077634. MDX-1105, also known as BMS-936559, is an anti-PD-L1antibody described in WO2007/005874. MEDI4736 is an anti-PD-L1monoclonal antibody described in WO2011/066389 and US2013/034559. In oneembodiment, the anti-PD-L1 antibody is atezolizumab.

In some embodiments, the therapeutic agent may comprise an antagonistdirected against CTLA-4 (also known as CD152), for example, a blockingantibody. In some embodiments, the therapeutic agent may compriseipilimumab (also known as MDX-010, MDX-101, or YERVOY®). In someembodiments, the therapeutic agent may comprise tremelimumab (also knownas ticilimumab or CP-675,206). In some embodiments, the therapeuticagent may comprise an antagonist directed against B7-H3 (also known asCD276), for example, a blocking antibody. In some embodiments, thetherapeutic agent may comprise MGA271. In some embodiments, thetherapeutic agent may comprise an antagonist directed against a TGFbeta, for example, metelimumab (also known as CAT-192), fresolimumab(also known as GC1008), or LY2157299.

In some embodiments, the therapeutic agent may comprise an agonistdirected against CD137 (also known as TNFRSF9, 4-1BB, or ILA), forexample, an activating antibody. In some embodiments, the therapeuticagent may comprise urelumab (also known as BMS-663513). In someembodiments, the therapeutic agent may comprise ligand of CD137 (alsoknown as TNFRSF9, 4-1BB, or ILA), such as 4-1BBL. In some embodiments,the therapeutic agent may comprise an agonist directed against CD40, forexample, an activating antibody. In some embodiments, the therapeuticagent may comprise CP-870893. In some embodiments, the therapeutic agentmay comprise an agonist directed against OX40 (also known as CD134), forexample, an activating antibody. In some embodiments, the therapeuticagent may comprise an anti-OX40 antibody (e.g., AgonOX). In someembodiments, the therapeutic agent may comprise a ligand of OX40, suchas OX40L. In some embodiments, the therapeutic agent may comprise anagonist directed against CD27, for example, an activating antibody. Insome embodiments, the therapeutic agent may comprise CDX-1127.

In some embodiments, the therapeutic agent may comprise a T cell (e.g.,a cytotoxic T cell or CTL) expressing a chimeric antigen receptor (CAR).In some embodiments, the therapeutic agent may comprise a T cellcomprising a dominant-negative TGF beta receptor, e.g, adominant-negative TGF beta type II receptor.

In some embodiments, the therapeutic agent may comprise an antibody-drugconjugate. In some embodiments, the antibody-drug conjugate comprisesmertansine or monomethyl auristatin E (MMAE). In some embodiments, thetherapeutic agent may comprise an anti-NaPi2b antibody-MMAE conjugate(also known as DNIB0600A or RG7599). In some embodiments, thetherapeutic agent may comprise trastuzumab emtansine (also known asT-DM1, ado-trastuzumab emtansine, or KADCYLA®). In some embodiments, thetherapeutic agent may comprise DMUC5754A. In some embodiments, thetherapeutic agent may comprise an antibody-drug conjugate targeting theendothelin B receptor (EDNBR), for example, an antibody directed againstEDNBR conjugated with MMAE (also known as DEDN6526A). In someembodiments, the therapeutic agent may comprise gemtuzumab ozogamicin(MYLOTARG®). In some embodiments, the therapeutic agent may compriseinotuzumab ozogamicin. In some embodiments, the therapeutic agent maycomprise bivatuzumab mertansine. In some embodiments, the therapeuticagent may comprise cantuzumab mertansine. In some embodiments, thetherapeutic agent may comprise cantuzumab ravtansine. In someembodiments, the therapeutic agent may comprise brentuximab vedotin(ADECTRIS®). In some embodiments, the therapeutic agent may comprisepinatuzumab vedotin. In some embodiments, the therapeutic agent maycomprise polatuzumab vedotin in some embodiments, the therapeutic agentmay comprise glembatumumab vedotin. In some embodiments, the therapeuticagent may comprise lorvotuzumab mertansine. In some embodiments, thetherapeutic agent may comprise tacatuzumab tetraxetan. In someembodiments, the therapeutic agent may comprise vandortuzumab vedotin(DSTP3086S). In some embodiments, the therapeutic agent may compriseibritumomab tiuxetan (ZEVALIN®)

In some embodiments, the therapeutic agent may comprise an antibodydirected against angiopoietin 2 (also known as Ang2). In someembodiments, the therapeutic agent may comprise MEDI3617.

In some embodiments, the therapeutic agent may comprise an antibodytargeting CSF-1R (also known as M-CSFR or CD115). In some embodiments,the therapeutic agent may comprise IMC-CS4 (LY3022855)). In someembodiments, the therapeutic agent may comprise emactuzumab.

In some embodiment, the therapeutic agent may comprise a cytokine. Insome embodiments, the therapeutic agent may comprise an interferon, forexample interferon alpha or interferon gamma. In some embodiments thetherapeutic agent may comprise Roferon-A (also known as recombinantInterferon alpha-2a). In some embodiments, the therapeutic agent maycomprise GM-CSF (also known as recombinant human granulocyte macrophagecolony stimulating factor, rhu GM-CSF, sargramostim, or LEUKIN E®). Insome embodiments, the therapeutic agent may comprise aldesleukin(PROLEUKIN®). In some embodiments, the therapeutic agent may compriseIL-12, In some embodiments, the therapeutic agent may comprise IL-10.

In some embodiments, the therapeutic agent may comprise an IL-2 fusionprotein. In some embodiments, the therapeutic agent may comprisetucotuzumab celmoleukin. In some embodiments, the therapeutic agent maycomprise darleukin. In some embodiments, the therapeutic agent maycomprise teleukin.

In some embodiments, the therapeutic agent may comprise an IL-10 fusionprotein. In some embodiments, the therapeutic agent may comprisedekavil. In some embodiments, the therapeutic agent may comprise a TNFfusion protein. In some embodiments, the therapeutic agent may comprisefibromun.

In some embodiments, the therapeutic agent may comprise a bispecificantibody. In some embodiments, the therapeutic agent may comprise abispecific antibody, such as, but not limited to, duligotuzumab, MM-111,MM141, TF2, ABT-981, ABT-122, LY3164530, SAR156597, GSK2434735,ozoralizumab, ALX-0761, ALX-0061, ALX-0141, ACE910.

In some embodiments, the therapeutic agent may comprise a bispecificantibody capable of binding to a T cell and a target cell, e.g. a tumorcell. In some embodiment, the therapeutic agent may comprise abispecific antibody that specifically binds to CD3 on a T cell and to atarget cell antigen. In some embodiment, the therapeutic agent maycomprise a bispecific T cell engager (BITE®). In some embodiments, thetherapeutic agent may comprise a bispecific antibody directed againstCD3 and CD19. In one embodiment, the bispecific antibody is blinatumomab(BLINCYTO®). In one embodiment, the bispecific antibody is AFM11. Insome embodiments, the therapeutic agent may comprise a bispecificantibody directed against CD3 and EpCAM. In one embodiment, thebispecific antibody is catumaxomab (REVOMAB®). In one embodiment, thebispecific antibody is solitomab (AMG 110, MT110). In some embodiments,the therapeutic agent may comprise a bispecific antibody directedagainst CD3 and Her2. In one embodiment, the bispecific antibody isertumaxomab. In some embodiments, the therapeutic agent may comprise abispecific antibody directed against CD3 and PSMA. In one embodiment,the bispecific antibody is BAY2010112 (AMG212, MT112). In someembodiments, the therapeutic agent may comprise a bispecific antibodydirected against CD3 and CEA. In one embodiment, the bispecific antibodyis MED1565 (AMG211, MT111). In some embodiments, the therapeutic agentmay comprise a bispecific antibody directed against CD3 and CD33. In oneembodiment, the bispecific antibody is AMG330. In some embodiments, thetherapeutic agent may comprise a bispecific antibody directed againstCD3 and CD123. In one embodiment, the bispecific antibody is MGD006. Inone embodiment, the bispecific antibody is XmAb® 14045. In someembodiments, the therapeutic agent may comprise a bispecific antibodydirected against CD3 and CD38. In some embodiments, the therapeuticagent may comprise a bispecific antibody directed against CD3 and gpA33.In one embodiment, the bispecific antibody is MGD007. In someembodiments, the therapeutic agent may comprise a bispecific antibodydirected against CD3 and CD20. In one embodiment, the bispecificantibody is XmAb® 13676. In one embodiment, the bispecific antibody isREGN1979. In one embodiment, the bispecific antibody is FBTA05(Lymphomun).

In some embodiments, the therapeutic agent may comprise a bispecificantibody directed against CD30 and CD16A. In one embodiment, thebispecific antibody is AFM13. In some embodiments, the therapeutic agentmay comprise a bispecific antibody directed against DR5 and FAP. In someembodiments, the therapeutic agent may comprise a bispecific antibodydirected against Ang2 and VEGF. In one embodiment, the bispecificantibody is vanucizumab.

In some embodiments, the therapeutic agent may comprise an Fc domain. Insome embodiments, the therapeutic agent may comprise a fusion proteincomprising an Fc domain.

In some embodiments, the therapeutic agent may comprise a recombinantreceptor or a fragment thereof. In some embodiments, the receptor is a Tcell receptor. In some embodiments, the receptor is a TNF receptor. Insome embodiments, the therapeutic agent may comprise etanercept(ENBREL®). In some embodiments, the receptor is a VEGF receptor. In someembodiments, the therapeutic agent may comprise ziv-aflibercept(ZALTRAP®). In some embodiments, the therapeutic agent may compriseaflibercept (EYLEA®). In some embodiments, the receptor is an IL-1receptor. In some embodiments, the therapeutic agent may compriserilonacept (ARCALYST®). In some embodiments, the therapeutic agent maycomprise IMCgp100. In some embodiments, the therapeutic agent maycomprise a chimeric antigen receptor (CAR). In some embodiments, thetherapeutic agent may comprise a Factor IX-Fc fusion protein. In someembodiments, the therapeutic agent may comprise a Factor VIII-Fc fusionprotein. In some embodiments, the therapeutic agent may comprise aCTLA-4-Fc fusion protein, such as e.g. belatacept, abatacept (ORENCIA®).In one embodiment, the therapeutic agent may comprise romiplostim.

In some embodiments, the therapeutic agent may comprise a recombinantreceptor ligand, such as a TNF receptor ligand.

In some embodiments, the therapeutic agent may comprise a generic,biosimilar or non-comparable biologic version of an agent, e.g. anantibody, named herein.

In one embodiment, the therapeutic agent does not comprise obinutuzumab.

T Cell Activating Therapeutic Agents

The following describes in further detail T cell activating therapeuticagents for which the invention may be useful, in particular aspects ofthe invention concerned with the reduction of cytokine releaseassociated with the administration of a therapeutic agent in a subject.

In some embodiments, the therapeutic agent comprises an antibody thatspecifically binds to an activating T cell antigen. In one embodiment,the therapeutic agent may comprise an antibody that specifically bindsto an antigen selected from the group of CD3, CD28, CD137 (also known as4-1BB), CD40, CD226, OX40, GITR, CD27, HVEM, and CD127.

In one embodiment, the therapeutic agent comprises an antibody thatspecifically binds to CD3, particularly CDR.

In one embodiment, the therapeutic agent comprises an antibody that isor can compete for binding with antibody H2C (PCT publication no.WO2008/119567), antibody V9 (Rodrigues et al., Int J Cancer Suppl 7,45-50 (1992) and U.S. Pat. No. 6,054,297), antibody FN18 (Nooij et al.,Eur J Immunol 19, 981-984 (1986)), antibody SP34 (Pessano et al., EMBO J4, 337-340 (1985)), antibody OKT3 (Kung et al., Science 206, 347-349(1979)), antibody WT31 (Spits et al., J Immunol 135, 1922 (1985)),antibody UCHT1 (Burns et al., J Immunol 129, 1451-1457 (1982)), antibody7D6 (Coulie et al., Eur J Immunol 21, 1703-1709 (1991)) or antibodyLeu-4. In some embodiments, the therapeutic agent may also comprise anantibody that specifically binds to CD3 as described in WO 2005/040220,WO 2005/118635, WO 2007/042261, WO 2008/119567, WO 2008/119565, WO2012/162067, WO 2013/158856, WO 2013/188693, WO 2013/186613, WO2014/110601, WO 2014/145806, WO 2014/191113, WO 2014/047231, WO2015/095392, WO 2015/181098, WO 2015/001085, WO 2015/104346, WO2015/172800, WO 2016/020444, or WO 2016/014974.

In one embodiment, the therapeutic agent may comprise an antibody thatspecifically binds to a B-cell antigen, particularly a malignant B-cellantigen. In one embodiment, the therapeutic agent may comprise anantibody that specifically binds to an antigen selected from the groupconsisting of CD20, CD19, CD22, ROR-1, CD37 and CD5, particularly toCD20 or CD19.

In some embodiments, the therapeutic agent may comprise an antibodyselected from rituximab, ocrelizumab, ofatumumab, ocaratuzumab,veltuzumab, and ublituximab.

In some embodiments, the therapeutic agent may comprise a multispecificantibody, particularly a bispecific antibody. In some embodiments, thetherapeutic agent may comprise a bispecific antibody capable of bindingto a T cell and a target cell, e.g. a tumor cell. In some embodiments,the target cell is a B-cell, particularly a malignant B-cell. In someembodiments, the therapeutic agent may comprise a bispecific antibodythat specifically binds to (i) an activating T cell antigen and (ii) a Bcell antigen. In some embodiments, the therapeutic agent may comprise abispecific antibody that specifically binds to CD3 on a T cell and to atarget cell antigen. In some embodiments, the target cell antigen is aB-cell antigen, particularly a malignant B-cell antigen. In someembodiments, the therapeutic agent may comprise a bispecific T cellengager (BiTE®).

In some embodiments, the therapeutic agent may comprise, a bispecificantibody directed against CD3 and CD20. In one embodiment, thebispecific antibody is XmAb® 13676. In one embodiment, the bispecificantibody is REGN1979. In one embodiment, the bispecific antibody isFBTA05 (Lymphomun).

In some embodiments, the therapeutic agent may comprise a bispecificantibody directed against CD3 and CD19. In one embodiment, thebispecific antibody is blinatumomab (BLINCYTO®). In one embodiment, thebispecific antibody is AFM1.1. In one embodiment, the bispecificantibody is MGD011 (JNJ-64052781).

In some embodiments, the therapeutic agent may comprise a bispecificantibody directed against CD3 and CD38. In one embodiment, thebispecific antibody is XmAb® 13551, XmAb® 15426, or XmAb® 14702.

In some embodiments, the therapeutic agent may comprise a bispecificantibody directed against CD3 and BCMA. In one embodiment, thebispecific antibody is BI836909.

In some embodiments, the therapeutic agent may comprise a bispecificantibody directed against CD3 and CD33. In one embodiment, thebispecific antibody is AMG330.

In some embodiments, the therapeutic agent may comprise a bispecificantibody directed against CD3 and CD123. In one embodiment, thebispecific antibody is MGD006. In one embodiment, the bispecificantibody is XmAb® 14045. In one embodiment, the bispecific antibody isJNJ-63709178.

In some embodiments, the therapeutic agent may comprise a recombinantreceptor or a fragment thereof. In some embodiments, the receptor is a Tcell receptor (TCR). In some embodiments, the therapeutic agent maycomprise a chimeric antigen receptor (CAR).

In some embodiments, the therapeutic agent may comprise a T cell (e.g.,a cytotoxic T cell or CTL) expressing a chimeric antigen receptor (CAR).In some embodiments, the therapeutic agent may comprise a T cellexpressing a recombinant T cell receptor (TCR).

In one embodiment, the therapeutic agent may comprise a CAR thatspecifically binds to a B-cell antigen, particularly a malignant B-cellantigen. In one embodiment, the therapeutic agent may comprise a CARthat specifically binds to an antigen selected from the group consistingof CD20, CD19, CD22, ROR-1, CD37 and CD5, particularly to CD20 or CD19.

In some embodiments, the therapeutic agent may comprise a CAR directedto CD19, or a T cell expressing a CAR directed to CD19. In someembodiments, the therapeutic agent may comprise KTE-C19, CTL019,JCAR-014, JCAR-015, JCAR-017, BPX-401, UCART19,

In some embodiments, the therapeutic agent may comprise a CAR directedto CD22, or a T cell expressing a CAR directed to CD22. In someembodiments, the therapeutic agent may comprise JCAR-018 or UCART22.

In some embodiments, the therapeutic agent may comprise an agonistdirected against an T cell activating co-stimulatory molecule. In someembodiments, a T cell activating co-stimulatory molecule may includeCD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In someembodiments, the agonist directed against a T cell activatingco-stimulatory molecule is an agonist antibody that binds to CD40,CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In someembodiments, the therapeutic agent may comprise an antibody targetingGITR. In some embodiments, the antibody targeting GITR is TRX518.

In some embodiments, the therapeutic agent may comprise an agonistdirected against CD137 (also known as TNFRSF9, 4-1BB, or ILA), forexample, an activating antibody. In some embodiments, the therapeuticagent may comprise urelumab (also known as BMS-663513). In someembodiments, the therapeutic agent may comprise ligand of CD137 (alsoknown as TNFRSF9, 4-1BB, or ILA), such as 4-1BBL. In some embodiments,the therapeutic agent may comprise an agonist directed against CD40, forexample, an activating antibody. In some embodiments, the therapeuticagent may comprise CP-870893. In some embodiments, the therapeutic agentmay comprise an agonist directed against OX40 (also known as CD134), forexample, an activating antibody. In some embodiments, the therapeuticagent may comprise an anti-OX40 antibody (e.g., AgonOX). In someembodiments, the therapeutic agent may comprise a ligand of OX40, suchas OX40L. In some embodiments, the therapeutic agent may comprise anagonist directed against CD27, for example, an activating antibody. Insome embodiments, the therapeutic agent may comprise CDX-1127.

Particular Therapeutic Agents

(i) Reduction of the Formation of Anti-Drug Antibodies (ADAs)

The therapeutic agents described in the following are particularlyuseful in the invention, in particular in relation aspects of theinvention concerned with the reduction of the formation of anti-drugantibodies (ADAs) against a therapeutic agent in a subject.

In some embodiments, the therapeutic agent comprises an antibody thatspecifically binds to carcinoembryonic antigen (CEA).

In one embodiment, the antibody that specifically binds to CEA comprisesa heavy chain variable region comprising the heavy chain CDR (HCDR) 1 ofSEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO:16; and a light chain variable region comprising the light chain CDR(LCDR) 1 of SEQ ID NO: 17, the LCDR2 of SEQ ID NO: 18 and the LCDR3 ofSEQ ID NO: 19. In a further embodiment, the antibody that specificallybinds CEA comprises a heavy chain variable region sequence that is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to of SEQ IDNO: 20 and a light chain variable region sequence that is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNO: 21. In a further embodiment, the antibody that specifically bindsCEA comprises the heavy chain variable region sequence of SEQ ID NO: 20and the light chain variable region sequence of SEQ ID NO: 21.

In one embodiment, the antibody that specifically binds to CEA comprisesa heavy chain variable region comprising the heavy chain CDR (HCDR) 1 ofSEQ ID NO: 136, the HCDR2 of SEQ ID NO: 137, and the HCDR3 of SEQ ID NO:138; and a light chain variable region comprising the light chain CDR(LCDR) 1 of SEQ ID NO: 139, the LCDR2 of SEQ ID NO: 140 and the LCDR3 ofSEQ ID NO: 141. In a further embodiment, the antibody that specificallybinds CEA comprises a heavy chain variable region sequence that is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to of SEQ IDNO: 142 and a light chain variable region sequence that is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNO: 143. In a further embodiment, the antibody that specifically bindsCEA comprises the heavy chain variable region sequence of SEQ ID NO: 142and the light chain variable region sequence of SEQ ID NO: 143.

In one embodiment, the antibody that specifically binds to CEA is afull-length antibody. In one embodiment, the antibody that specificallybinds to CEA is an antibody of the human IgG class, particularly anantibody of the human IgG₁ class. In one embodiment, the antibody thatspecifically binds to CEA is an antibody fragment, particularly a Fabmolecule or a scFv molecule, more particularly a Fab molecule. In oneembodiment, the antibody that specifically binds to CEA is a humanizedantibody.

In some embodiments, the therapeutic agent comprises an antibody thatspecifically binds to fibroblast activation protein (FAP). In oneembodiment, the antibody that specifically binds FAP comprises a heavychain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to of SEQ ID NO: 25 and a light chainvariable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 26. In a furtherembodiment, the antibody that specifically binds FAP comprises the heavychain variable region sequence of SEQ ID NO: 25 and the light chainvariable region sequence of SEQ ID NO: 26.

In one embodiment, the antibody that specifically binds to FAP is afull-length antibody. In one embodiment, the antibody that specificallybinds to FAP is an antibody of the human IgG class, particularly anantibody of the human IgG₁ class. In one embodiment, the antibody thatspecifically binds to FAP is an antibody fragment, particularly a Fabmolecule or a scFv molecule, more particularly a Fab molecule. In oneembodiment, the antibody that specifically binds to FAP is a humanantibody.

In some embodiments, the therapeutic agent comprises an antibody thatspecifically binds to CD3, particularly CD3 epsilon. In one embodiment,the antibody that specifically binds to CD3 comprises a heavy chainvariable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO:32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and alight chain variable region comprising the light chain CDR (LCDR) 1 ofSEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO:37. In a further embodiment, the antibody that specifically binds CD3comprises a heavy chain variable region sequence that is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to of SEQ ID NO: 38 and alight chain variable region sequence that is at least 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 39.In a further embodiment, the antibody that specifically binds CD3comprises the heavy chain variable region sequence of SEQ ID NO: 38 andthe light chain variable region sequence of SEQ ID NO: 39.

In one embodiment, the antibody that specifically binds to CD3 is afull-length antibody. In one embodiment, the antibody that specificallybinds to CD3 is an antibody of the human IgG class, particularly anantibody of the human IgG₁ class. In one embodiment, the antibody thatspecifically binds to CD3 is an antibody fragment, particularly a Fabmolecule or a scFv molecule, more particularly a Fab molecule. In aparticular embodiment, the antibody that specifically binds to CD3 is acrossover Fab molecule wherein the variable domains or the constantdomains of the Fab heavy and light chain are exchanged (i.e. replaced byeach other). In one embodiment, the antibody that specifically binds toCD3 is a humanized antibody.

In some embodiments, the therapeutic agent comprises a cytokine. In oneembodiment the cytokine is selected from the group consisting of,GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10,IL-12, IL-15, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, andTNF-β. In one embodiment, the cytokine is IL-2, particularly human IL-2.The sequence of wild-type human IL-2 is shown in SEQ ID NO: 12.

In one embodiment, the therapeutic agent comprises a mutant IL-2polypeptide having reduced binding affinity to the α-subunit of the IL-2receptor as compared to wild-type IL-2. Together with the β- andγ-subunits (also known as CD122 and CD132, respectively), the α-subunit(also known as CD25) forms the heterotrimeric high-affinity IL-2receptor, while the dimeric receptor consisting only of the β- andγ-subunits is termed the intermediate-affinity IL-2 receptor. A mutantIL-2 polypeptide with reduced binding to the α-subunit of the IL-2receptor has a reduced ability to induce IL-2 signaling in regulatory T(T_(reg)) cells, induces less activation-induced cell death (AICD) in Tcells, and has a reduced toxicity profile in vivo, compared to awild-type IL-2 polypeptide (see e.g. WO 2012/107417, incorporated hereinby reference in its entirety).

In a more specific embodiment, the mutant IL-2 polypeptide comprisesthree amino acid substitutions at the positions corresponding to residue42, 45 and 72 of human IL-2. In an even more specific embodiment, themutant IL-2 polypeptide is a human IL-2 polypeptide comprising the aminoacid substitutions F42A, Y45A and L72G (numbering relative to the humanIL-2 sequence SEQ ID NO: 12). In one embodiment the mutant IL-2polypeptide additionally comprises an amino acid mutation at a positioncorresponding to position 3 of human IL-2, which eliminates theO-glycosylation site of IL-2. In one embodiment said amino acid mutationwhich eliminates the O-glycosylation site of IL-2 at a positioncorresponding to residue 3 of human IL-2 is an amino acid substitutionselected from the group of T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, andT3P. Particularly, said additional amino acid mutation is an amino acidsubstitution replacing a threonine residue by an alanine residue. Aparticular mutant IL-2 polypeptide useful in the invention comprisesfour amino acid substitutions at positions corresponding to residues 3,42, 45 and 72 of human IL-2. Specific amino acid substitutions are T3A,F42A, Y45A and L72G. This mutant IL-2 polypeptide exhibits no detectablebinding to CD25, reduced ability to induce apoptosis in T cells, reducedability to induce IL-2 signaling in T_(re)g cells, and a reducedtoxicity profile in vivo (see e.g. WO 2012/107417, incorporated hereinby reference in its entirety). However, it retains ability to activateIL-2 signaling in effector cells, to induce proliferation of effectorcells, and to generate IFN-γ as a secondary cytokine by NK cells.

The IL-2 or mutant IL-2 polypeptide according to any of the aboveembodiments may comprise additional mutations that provide furtheradvantages such as increased expression or stability. For example, thecysteine at position 125 may be replaced with a neutral amino acid suchas serine, alanine, threonine or valine, yielding C125S IL-2, C125AIL-2, C125T IL-2 or C125V IL-2 respectively, as described in U.S. Pat.No. 4,518,584. As described therein, one may also delete the N-terminalalanine residue of IL-2 yielding such mutants as des-A1 C125S or des-A1C125A. Alternatively or conjunctively, the IL-2 mutant may include amutation whereby methionine normally occurring at position 104 ofwild-type human IL-2 is replaced by a neutral amino acid such as alanine(see U.S. Pat. No. 5,206,344). The resulting mutants, e. g., des-A1M104A IL-2, des-A1 M104A C125S IL-2, M104A IL-2, M104A C125A IL-2,des-A1 M104A C125A IL-2, or M104A C125S IL-2 (these and other mutantsmay be found in U.S. Pat. No. 5,116,943 and in Weiger et al., Eur JBiochem 180, 295-300 (1989)) may be used in conjunction with theparticular IL-2 mutations described herein.

Thus, in certain embodiments the IL-2 or mutant IL-2 polypeptidecomprises an additional amino acid mutation at a position correspondingto residue 125 of human IL-2. In one embodiment said additional aminoacid mutation is the amino acid substitution C125A. In certainembodiments the mutant IL-2 polypeptide is essentially a full-lengthIL-2 molecule, particularly a human full-length IL-2 molecule. In oneembodiment, the mutant IL-2 polypeptide comprises a polypeptide sequencethat is at least 80%, at least 85%, or at least 90% identical to thesequence of SEQ ID NO: 12.

In a specific embodiment the mutant IL-2 polypeptide comprises thepolypeptide sequence of SEQ ID NO: 13.

In some embodiments, the therapeutic agent comprises an immunoconjugate.Particular immunoconjugates are described in WO 2012/107417 and WO2012/146628 (each incorporated herein by reference in its entirety).

In one embodiment, the immunoconjugate comprises an antibody thatspecifically binds to CEA as described herein, and a mutant IL-2polypeptide as described herein. In one embodiment, the antibody is afull-length antibody.

In one embodiment the therapeutic agent comprises an immunoconjugatecomprising

-   -   (i) an antibody of the human IgG₁ subclass that specifically        binds to CEA and comprises a heavy chain variable region        comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 14, the        HCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO: 16; and a        light chain variable region comprising the light chain CDR        (LCDR) 1 of SEQ ID NO: 17, the LCDR2 of SEQ ID NO: 18 and the        LCDR3 of SEQ ID NO: 19; and    -   (ii) a mutant human IL-2 polypeptide comprising the amino acid        substitutions F42A, Y45A and L72G (numbering relative to the        human IL-2 sequence SEQ ID NO: 12).

In one embodiment, the immunoconjugate comprises an antibody thatspecifically binds to FAP as described herein, and a mutant IL-2polypeptide as described herein. In one embodiment, the antibody is afull-length antibody.

In one embodiment the therapeutic agent comprises an immunoconjugatecomprising

-   -   (i) an antibody of the human IgG₁ subclass that specifically        binds to FAP and comprises the heavy chain variable region of        SEQ ID NO: 25; and the light chain variable region of SEQ ID NO:        26; and    -   (ii) a mutant human IL-2 polypeptide comprising the amino acid        substitutions F42A, Y45A and L72G (numbering relative to the        human IL-2 sequence SEQ ID NO: 12).

In one embodiment, the immunoconjugate comprises no more than one mutantIL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide isfused to the carboxy-terminal amino acid of one of the antibody heavychains, optionally through a linker peptide. Suitable, non-immunogeniclinker peptides include, for example, (G₄S)_(n), (SG₄)_(n) orG₄(SG₄)_(n) linker peptides, wherein n is generally a number between 1and 10, typically between 2 and 4. In one embodiment, the linker peptideis (G₄S)₃.

In one embodiment, the immunoconjugate comprises a polypeptidecomprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 22, a polypeptidecomprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 23, and apolypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 24.

In one embodiment, the immunoconjugate comprises a polypeptidecomprising the sequence of SEQ ID NO: 22, a polypeptide comprising thesequence of SEQ ID NO: 23, and a polypeptide comprising the sequence ofSEQ ID NO: 24.

In one embodiment, the immunoconjugate is cergutuzumab amunaleukin (seeWHO Drug Information (International Nonproprietary Names forPharmaceutical Substances), Recommended INN: List 75, 2016,pre-publication copy” (incorporated herein by reference in itsentirety).

In one embodiment, the immunoconjugate comprises a polypeptidecomprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 27, a polypeptidecomprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 28, and apolypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 29.

In one embodiment, the immunoconjugate comprises a polypeptidecomprising the sequence of SEQ ID NO: 27, a polypeptide comprising thesequence of SEQ ID NO: 28, and a polypeptide comprising the sequence ofSEQ ID NO: 29.

In one embodiment, the therapeutic agent comprises a bispecificantibody. Particular bispecific antibodies are described in PCTpublication nos. WO 2013/026833 and WO 2014/131712 and in PCTapplication no. PCT/EP2016/073171 (each incorporated herein by referencein its entirety).

In one embodiment, the bispecific antibody comprises an antibody thatspecifically binds to CEA as described herein, and an antibody thatspecifically binds to CD3 as described herein.

In one embodiment, the bispecific antibody comprises a first antibodythat specifically binds to CD3 as described herein, and a second and athird antibody that specifically bind to CEA as described herein. In oneembodiment, the first antibody is a crossover Fab molecule as describedherein, and the second and the first antibody are each a conventionalFab molecule. In one embodiment, the bispecific antibody furthercomprises an Fc domain as described herein. The bispecific antibody mayhave the antibody formats described herein and may comprise the antigenbinding moieties described herein. The bispecific antibody may comprisemodifications in the Fc region and/or the antigen binding moieties asdescribed herein.

In one embodiment the therapeutic agent comprises a bispecific antibodycomprising

-   -   (i) a first antigen binding moiety that specifically binds to        CD3, comprising a heavy chain variable region comprising the        heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID        NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain        variable region comprising the light chain CDR (LCDR) 1 of SEQ        ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID        NO: 37, wherein the first antigen binding moiety is a crossover        Fab molecule wherein either the variable or the constant        regions, particularly the constant regions, of the Fab light        chain and the Fab heavy chain are exchanged;    -   (ii) a second and a third antigen binding moiety that        specifically bind to CEA, comprising a heavy chain variable        region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 14,        the HCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO: 16; and        a light chain variable region comprising the light chain CDR        (LCDR) 1 of SEQ ID NO: 17, the LCDR2 of SEQ ID NO: 18 and the        LCDR3 of SEQ ID NO: 19, wherein the second and third antigen        binding moiety are each a Fab molecule, particularly a        conventional Fab molecule;    -   (iii) an Fc domain composed of a first and a second subunit        capable of stable association, wherein the second antigen        binding moiety is fused at the C-terminus of the Fab heavy chain        to the N-terminus of the Fab heavy chain of the first antigen        binding moiety, and the first antigen binding moiety is fused at        the C-terminus of the Fab heavy chain to the N-terminus of the        first subunit of the Fc domain, and wherein the third antigen        binding moiety is fused at the C-terminus of the Fab heavy chain        to the N-terminus of the second subunit of the Fc domain.

In one embodiment, the first antigen binding moiety that specificallybinds to CD3, comprises the heavy chain variable region of SEQ ID NO:38, and the light chain variable region of SEQ ID NO: 39. In oneembodiment, the second and third antigen binding moieties thatspecifically bind to CEA comprise the heavy chain variable region of SEQID NO: 20, and the light chain variable region of SEQ ID NO: 21.

In one embodiment, the antigen binding moieties and the Fc region arefused to each other by peptide linkers, particularly by peptide linkersas in SEQ ID NO: 42 and SEQ ID NO: 43. In one embodiment, the bispecificantibody comprises a polypeptide comprising a sequence that is at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 40, a polypeptide comprising a sequence that is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNO: 41, a polypeptide comprising a sequence that is at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:42, and a polypeptide comprising a sequence that is at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:43.

In one embodiment, the bispecific antibody comprises a polypeptidecomprising the sequence of SEQ ID NO: 40, a polypeptide comprising thesequence of SEQ ID NO: 41, a polypeptide comprising the sequence of SEQID NO: 42, and a polypeptide comprising the sequence of SEQ ID NO: 43.(CEA TCB)

In one embodiment the therapeutic antibody comprises a bispecificantibody comprising

-   -   (i) a first antigen binding moiety that specifically binds to        CD3, comprising a heavy chain variable region comprising the        heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID        NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain        variable region comprising the light chain CDR (LCDR) 1 of SEQ        ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID        NO: 37, wherein the first antigen binding moiety is a crossover        Fab molecule wherein either the variable or the constant        regions, particularly the variable regions, of the Fab light        chain and the Fab heavy chain are exchanged;    -   (ii) a second and a third antigen binding moiety that        specifically bind to CEA, comprising a heavy chain variable        region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO:        136, the HCDR2 of SEQ ID NO: 137, and the HCDR3 of SEQ ID NO:        138; and a light chain variable region comprising the light        chain CDR (LCDR) 1 of SEQ ID NO: 139, the LCDR2 of SEQ ID NO:        140 and the LCDR3 of SEQ ID NO: 141, wherein the second and        third antigen binding moiety are each a Fab molecule,        particularly a conventional Fab molecule;    -   (iii) an Fc domain composed of a first and a second subunit        capable of stable association, wherein the second antigen        binding moiety is fused at the C-terminus of the Fab heavy chain        to the N-terminus of the Fab heavy chain of the first antigen        binding moiety, and the first antigen binding moiety is fused at        the C-terminus of the Fab heavy chain to the N-terminus of the        first subunit of the Fc domain, and wherein the third antigen        binding moiety is fused at the C-terminus of the Fab heavy chain        to the N-terminus of the second subunit of the Fc domain.

In one embodiment, the first antigen binding moiety that specificallybinds to CD3, comprises the heavy chain variable region of SEQ ID NO:38, and the light chain variable region of SEQ ID NO: 39. In oneembodiment, the second and third antigen binding moiety thatspecifically bind to CEA comprise the heavy chain variable region of SEQID NO: 142, and the light chain variable region of SEQ ID NO: 143.

In one embodiment, the antigen binding moieties and the Fc region arefused to each other by peptide linkers, particularly by peptide linkersas in SEQ ID NO: 145 and SEQ ID NO: 146.

In one embodiment, in the constant domain CL of the second and the thirdFab molecule under (ii) the amino acid at position 124 is substituted bylysine (K) (numbering according to Kabat) and the amino acid at position123 is substituted by lysine (K) or arginine (R), particularly byarginine (R) (numbering according to Kabat), and in the constant domainCH1 of the second and the third Fab molecule under (ii) the amino acidat position 147 is substituted by glutamic acid (E) (numbering accordingto Kabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index).

In one embodiment, the bispecific antibody comprises a polypeptidecomprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 144, a polypeptidecomprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 145, a polypeptidecomprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 146, and apolypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 147.

In one embodiment, the bispecific antibody comprises a polypeptidecomprising the sequence of SEQ ID NO: 144, a polypeptide comprising thesequence of SEQ ID NO: 145, a polypeptide comprising the sequence of SEQID NO: 146, and a polypeptide comprising the sequence of SEQ ID NO: 147.

(ii) Reduction of Cytokine Release

The therapeutic agents described in the following are particularlyuseful in the invention, in particular in relation aspects of theinvention concerned with the reduction of cytokine release associatedwith the administration of a therapeutic agent in a subject.

The aspects of the invention concerned with the reduction of cytokinerelease associated with the administration of a therapeutic agent in asubject are particularly useful in connection with therapeutic agentsthat are activating T-cells in the subject (T cell activatingtherapeutic agents), i.e. have the ability of inducing T-cell activationin the subject. Such therapeutic agents include, for example, antibodiesdirected to T-cell antigens (particularly activating T-cell antigens),or T-cells modified with chimeric antigen receptors (CAR) or recombinantT-cell receptors (TCR). The aspects of the invention concerned with thereduction of cytokine release associated with the administration of atherapeutic agent in a subject are particularly useful in connectionwith B-cell targeted T-cell activating therapeutic agents.

In some embodiments, the therapeutic agent comprises an antibody thatspecifically binds to CD3, particularly CD3 epsilon.

In one embodiment, the antibody that specifically binds to CD3 comprisesa heavy chain variable region comprising the heavy chain CDR (HCDR) 1 ofSEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO:34; and a light chain variable region comprising the light chain CDR(LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 ofSEQ ID NO: 37. In a further embodiment, the antibody that specificallybinds CD3 comprises a heavy chain variable region sequence that is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to of SEQ IDNO: 38 and a light chain variable region sequence that is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNO: 39. In a further embodiment, the antibody that specifically bindsCD3 comprises the heavy chain variable region sequence of SEQ ID NO: 38and the light chain variable region sequence of SEQ ID NO: 39.

In one embodiment, the antibody that specifically binds to CD3 comprisesa heavy chain variable region comprising the heavy chain HVR 1 (H1-HVR)of SEQ ID NO: 120, the H2-HVR of SEQ ID NO: 121, and the H3-HVR of SEQID NO: 122; and a light chain variable region comprising the light chainHVR 1 (L1-HVR) of SEQ ID NO: 123, the L2-HVR of SEQ ID NO: 124 and theL3-HVR of SEQ ID NO: 125. In a further embodiment, the antibody thatspecifically binds CD3 comprises a heavy chain variable region sequencethat is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toof SEQ ID NO: 126 and a light chain variable region sequence that is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 127. In a further embodiment, the antibody thatspecifically binds CD3 comprises the heavy chain variable regionsequence of SEQ ID NO: 126 and the light chain variable region sequenceof SEQ ID NO: 127.

In one embodiment, the antibody that specifically binds to CD3 is afull-length antibody. In one embodiment, the antibody that specificallybinds to CD3 is an antibody of the human IgG class, particularly anantibody of the human IgG₁ class. In one embodiment, the antibody thatspecifically binds to CD3 is an antibody fragment, particularly a Fabmolecule or a scFv molecule, more particularly a Fab molecule. In aparticular embodiment, the antibody that specifically binds to CD3 is acrossover Fab molecule wherein the variable domains or the constantdomains of the Fab heavy and light chain are exchanged (i.e. replaced byeach other). In one embodiment, the antibody that specifically binds toCD3 is a humanized antibody.

In one embodiment, the therapeutic agent comprises a multispecificantibody, particularly a bispecific antibody. In one embodiment, themultispecific antibody specifically binds to (i) an activating T cellantigen and (ii) a B cell antigen. Particular bispecific antibodies aredescribed in PCT publication no. WO 2016/020309 and PCT application no.PCT/EP2016/073041, as well as PCT publication no. WO 2015/095392 (eachincorporated herein by reference in its entirety).

In one embodiment, the bispecific antibody specifically binds to CD3 andCD20. In one embodiment, the bispecific antibody comprises an antigenbinding moiety that specifically binds to CD20, and an antigen bindingmoiety that specifically binds to CD3. In one embodiment, the bispecificantibody comprises a first antigen binding moiety that specificallybinds to CD3, and a second and a third antigen binding moiety thatspecifically bind to CD20. In one embodiment, the first antigen bindingmoiety is a crossover Fab molecule, and the second and the first antigenbinding moiety are each a conventional Fab molecule. In one embodiment,the bispecific antibody further comprises an Fc domain. The bispecificantibody may have the antibody formats described herein and may comprisethe antigen binding moieties described herein. The bispecific antibodymay comprise modifications in the Fc region and/or the antigen bindingmoieties as described herein.

In one embodiment, the therapeutic agent comprises a bispecific antibodycomprising

-   -   (i) an antigen binding moiety that specifically binds to CD3 and        comprises a heavy chain variable region comprising the heavy        chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33,        and the HCDR3 of SEQ ID NO: 34; and a light chain variable        region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35,        the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37; and    -   (ii) an antigen binding moiety that specifically binds to CD20        and comprises a heavy chain variable region comprising the heavy        chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5,        and the HCDR3 of SEQ ID NO: 6; and a light chain variable region        comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the        LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.

In one embodiment, the therapeutic agent comprises a bispecific antibodycomprising

-   -   (i) an antigen binding moiety that specifically binds to CD3 and        comprises a heavy chain variable region of SEQ ID NO: 38; and a        light chain variable region of SEQ ID NO: 39; and    -   (ii) an antigen binding moiety that specifically binds to CD20        and comprises a heavy chain variable region of SEQ ID NO: 10;        and a light chain variable region of SEQ ID NO: 11.

In a particular embodiment, the therapeutic agent comprises a bispecificantibody comprising

-   -   a) a first Fab molecule which specifically binds to a first        antigen;    -   b) a second Fab molecule which specifically binds to a second        antigen, and wherein the variable domains VL and VH of the Fab        light chain and the Fab heavy chain are replaced by each other;    -   c) a third Fab molecule which specifically binds to the first        antigen; and    -   d) an Fc domain composed of a first and a second subunit capable        of stable association;    -   wherein    -   (i) the first antigen is CD20 and the second antigen is CD3,        particularly CD3 epsilon;    -   (ii) the first Fab molecule under a) and the third Fab molecule        under c) each comprise the heavy chain complementarity        determining region (CDR) 1 of SEQ ID NO: 4, the heavy chain CDR        2 of SEQ ID NO: 5, the heavy chain CDR 3 of SEQ ID NO: 6, the        light chain CDR 1 of SEQ ID NO: 7, the light chain CDR 2 of SEQ        ID NO: 8 and the light chain CDR 3 of SEQ ID NO: 9, and the        second Fab molecule under b) comprises the heavy chain CDR 1 of        SEQ ID NO: 32, the heavy chain CDR 2 of SEQ ID NO: 33, the heavy        chain CDR 3 of SEQ ID NO: 34, the light chain CDR 1 of SEQ ID        NO: 35, the light chain CDR 2 of SEQ ID NO: 36 and the light        chain CDR 3 of SEQ ID NO: 37;    -   (iii) in the constant domain CL of the first Fab molecule        under a) and the third Fab molecule under c) the amino acid at        position 124 is substituted by lysine (K) (numbering according        to Kabat) and the amino acid at position 123 is substituted by        lysine (K) or arginine (R), particularly by arginine (R)        (numbering according to Kabat), and wherein in the constant        domain CH1 of the first Fab molecule under a) and the third Fab        molecule under c) the amino acid at position 147 is substituted        by glutamic acid (E) (numbering according to Kabat EU index) and        the amino acid at position 213 is substituted by glutamic        acid (E) (numbering according to Kabat EU index); and    -   (iv) the first Fab molecule under a) is fused at the C-terminus        of the Fab heavy chain to the N-terminus of the Fab heavy chain        of the second Fab molecule under b), and the second Fab molecule        under b) and the third Fab molecule under c) are each fused at        the C-terminus of the Fab heavy chain to the N-terminus of one        of the subunits of the Fc domain under d).

In one embodiment, the first Fab molecule under a) and the third Fabmolecule under c) each comprise a heavy chain variable region that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:10, and a light chain variable region that is at least 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 11.

In one embodiment, the first Fab molecule under a) and the third Fabmolecule under c) each comprise the heavy chain variable region sequenceof SEQ ID NO: 10, and the light chain variable region sequence of SEQ IDNO: 11.

In one embodiment, the second Fab molecule under b) comprises a heavychain variable region that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 38, and a light chain variableregion that is at least 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 39.

In still a further embodiment, the second Fab molecule under b)comprises the heavy chain variable region sequence of SEQ ID NO: 38, andthe light chain variable region sequence of SEQ ID NO: 39.

In a particular embodiment, the bispecific antibody comprises apolypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 44, a polypeptide that is at least 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 45, a polypeptidethat is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 46, and a polypeptide that is at least 95%, 96%, 97%, 98%, or99% identical to the sequence of SEQ ID NO: 47. In a further particularembodiment, the bispecific antibody comprises a polypeptide sequence ofSEQ ID NO: 44, a polypeptide sequence of SEQ ID NO: 45, a polypeptidesequence of SEQ ID NO: 46 and a polypeptide sequence of SEQ ID NO: 47.(CD20×CD3 bsAB)

In one embodiment, the therapeutic agent comprises a bispecific antibodycomprising

-   -   (i) an antigen binding moiety that specifically binds to CD3 and        comprises a heavy chain variable region comprising the heavy        chain HVR 1 (H1-HVR) of SEQ ID NO: 120, the H2-HVR of SEQ ID NO:        121, and the H3-HVR of SEQ ID NO: 122; and a light chain        variable region comprising the light chain HVR 1 (L1-HVR) of SEQ        ID NO: 123, the L2-HVR of SEQ ID NO: 124 and the L3-HVR of SEQ        ID NO: 125; and    -   (ii) an antigen binding moiety that specifically binds to CD20        and comprises a heavy chain variable region comprising the heavy        chain HVR 1 (H1-HVR) of SEQ ID NO: 128, the H2-HVR of SEQ ID NO:        129, and the H3-HVR of SEQ ID NO: 130; and a light chain        variable region comprising the light chain HVR 1 (L1-HVR) of SEQ        ID NO: 131, the L2-HVR of SEQ ID NO: 132 and the L3-HVR of SEQ        ID NO: 133.

In one embodiment, the therapeutic agent comprises a bispecific antibodycomprising

-   -   (i) an antigen binding moiety that specifically binds to CD3 and        comprises a heavy chain variable region of SEQ ID NO: 126; and a        light chain variable region of SEQ ID NO: 127; and    -   (ii) an antigen binding moiety that specifically binds to CD20        and comprises a heavy chain variable region of SEQ ID NO: 134;        and a light chain variable region of SEQ ID NO: 135.

In one embodiment, the bispecific antibody comprises an antigen bindingmoiety that specifically binds to CD19, and an antigen binding moietythat specifically binds to CD3. In one embodiment, the bispecificantibody comprises a first antigen binding moiety that specificallybinds to CD3, and a second and a third antigen binding moiety thatspecifically bind to CD19. In one embodiment, the first antigen bindingmoiety is a crossover Fab molecule, and the second and the first antigenbinding moiety are each a conventional Fab molecule. In one embodiment,the bispecific antibody further comprises an Fc domain. The bispecificantibody may comprise modifications in the Fc region and/or the antigenbinding moieties as described herein.

In one embodiment, the therapeutic agent comprises a bispecific antibodycomprising

-   -   (i) an antigen binding moiety that specifically binds to CD3 and        comprises a heavy chain variable region comprising the heavy        chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33,        and the HCDR3 of SEQ ID NO: 34; and a light chain variable        region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35,        the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37; and    -   (ii) an antigen binding moiety that specifically binds to CD19        and comprises a heavy chain variable region comprising the heavy        chain CDR (HCDR) 1 of SEQ ID NO: 48, the HCDR2 of SEQ ID NO: 49,        and the HCDR3 of SEQ ID NO: 50; and a light chain variable        region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 51,        the LCDR2 of SEQ ID NO: 52 and the LCDR3 of SEQ ID NO: 53.

In one embodiment, the therapeutic agent comprises a bispecific antibodycomprising

-   -   (i) an antigen binding moiety that specifically binds to CD3 and        comprises a heavy chain variable region of SEQ ID NO: 38; and a        light chain variable region of SEQ ID NO: 39; and    -   (ii) an antigen binding moiety that specifically binds to CD19        and comprises a heavy chain variable region of SEQ ID NO: 54;        and a light chain variable region of SEQ ID NO: 55.

In a particular embodiment, the therapeutic agent comprises a bispecificantibody comprising

-   -   a) a first Fab molecule which specifically binds to a first        antigen;    -   b) a second Fab molecule which specifically binds to a second        antigen, and wherein the variable domains VL and VH of the Fab        light chain and the Fab heavy chain are replaced by each other;    -   c) a third Fab molecule which specifically binds to the first        antigen; and    -   d) an Fc domain composed of a first and a second subunit capable        of stable association;    -   wherein    -   (i) the first antigen is CD19 and the second antigen is CD3,        particularly CD3 epsilon;    -   (ii) the first Fab molecule under a) and the third Fab molecule        under c) each comprise the heavy chain complementarity        determining region (CDR) 1 of SEQ ID NO: 48, the heavy chain CDR        2 of SEQ ID NO: 49, the heavy chain CDR 3 of SEQ ID NO: 50, the        light chain CDR 1 of SEQ ID NO: 51, the light chain CDR 2 of SEQ        ID NO: 52 and the light chain CDR 3 of SEQ ID NO: 53, and the        second Fab molecule under b) comprises the heavy chain CDR 1 of        SEQ ID NO: 32, the heavy chain CDR 2 of SEQ ID NO: 33, the heavy        chain CDR 3 of SEQ ID NO: 34, the light chain CDR 1 of SEQ ID        NO: 35, the light chain CDR 2 of SEQ ID NO: 36 and the light        chain CDR 3 of SEQ ID NO: 37;    -   (iii) in the constant domain CL of the first Fab molecule        under a) and the third Fab molecule under c) the amino acid at        position 124 is substituted by lysine (K) (numbering according        to Kabat) and the amino acid at position 123 is substituted by        lysine (K) or arginine (R), particularly by arginine (R)        (numbering according to Kabat), and wherein in the constant        domain CH1 of the first Fab molecule under a) and the third Fab        molecule under c) the amino acid at position 147 is substituted        by glutamic acid (E) (numbering according to Kabat EU index) and        the amino acid at position 213 is substituted by glutamic        acid (E) (numbering according to Kabat EU index); and    -   (iv) the first Fab molecule under a) is fused at the C-terminus        of the Fab heavy chain to the N-terminus of the Fab heavy chain        of the second Fab molecule under b), and the second Fab molecule        under b) and the third Fab molecule under c) are each fused at        the C-terminus of the Fab heavy chain to the N-terminus of one        of the subunits of the Fc domain under d).

In one embodiment, the first Fab molecule under a) and the third Fabmolecule under c) each comprise a heavy chain variable region that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:54, and a light chain variable region that is at least 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 55.

In one embodiment, the first Fab molecule under a) and the third Fabmolecule under c) each comprise the heavy chain variable region sequenceof SEQ ID NO: 54, and the light chain variable region sequence of SEQ IDNO: 55.

In one embodiment, the second Fab molecule under b) comprises a heavychain variable region that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 38, and a light chain variableregion that is at least 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 39.

In still a further embodiment, the second Fab molecule under b)comprises the heavy chain variable region sequence of SEQ ID NO: 38, andthe light chain variable region sequence of SEQ ID NO: 39.

In a particular embodiment, the bispecific antibody comprises apolypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 47, a polypeptide that is at least 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 56, a polypeptidethat is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 57, and a polypeptide that is at least 95%, 96%, 97%, 98%, or99% identical to the sequence of SEQ ID NO: 58. In a further particularembodiment, the bispecific antibody comprises a polypeptide sequence ofSEQ ID NO: 47, a polypeptide sequence of SEQ ID NO: 56, a polypeptidesequence of SEQ ID NO: 57 and a polypeptide sequence of SEQ ID NO: 58.

In one embodiment, the therapeutic agent comprises a bispecific antibodycomprising

-   -   (i) an antigen binding moiety that specifically binds to CD3 and        comprises a heavy chain variable region comprising the heavy        chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33,        and the HCDR3 of SEQ ID NO: 34; and a light chain variable        region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35,        the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37; and    -   (ii) an antigen binding moiety that specifically binds to CD19        and comprises a heavy chain variable region comprising the heavy        chain CDR (HCDR) 1 of SEQ ID NO: 59, the HCDR2 of SEQ ID NO: 60,        and the HCDR3 of SEQ ID NO: 61; and a light chain variable        region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 62,        the LCDR2 of SEQ ID NO: 63 and the LCDR3 of SEQ ID NO: 64.

In one embodiment, the therapeutic agent comprises a bispecific antibodycomprising

-   -   (i) an antigen binding moiety that specifically binds to CD3 and        comprises a heavy chain variable region of SEQ ID NO: 38; and a        light chain variable region of SEQ ID NO: 39; and    -   (ii) an antigen binding moiety that specifically binds to CD19        and comprises a heavy chain variable region of SEQ ID NO: 65;        and a light chain variable region of SEQ ID NO: 66.

In a particular embodiment, the therapeutic agent comprises a bispecificantibody comprising

-   -   a) a first Fab molecule which specifically binds to a first        antigen;    -   b) a second Fab molecule which specifically binds to a second        antigen, and wherein the variable domains VL and VH of the Fab        light chain and the Fab heavy chain are replaced by each other;    -   c) a third Fab molecule which specifically binds to the first        antigen; and    -   d) an Fc domain composed of a first and a second subunit capable        of stable association;    -   wherein    -   (i) the first antigen is CD19 and the second antigen is CD3,        particularly CD3 epsilon;    -   (ii) the first Fab molecule under a) and the third Fab molecule        under c) each comprise the heavy chain complementarity        determining region (CDR) 1 of SEQ ID NO: 59, the heavy chain CDR        2 of SEQ ID NO: 60, the heavy chain CDR 3 of SEQ ID NO: 61, the        light chain CDR 1 of SEQ ID NO: 62, the light chain CDR 2 of SEQ        ID NO: 63 and the light chain CDR 3 of SEQ ID NO: 64, and the        second Fab molecule under b) comprises the heavy chain CDR 1 of        SEQ ID NO: 32, the heavy chain CDR 2 of SEQ ID NO: 33, the heavy        chain CDR 3 of SEQ ID NO: 34, the light chain CDR 1 of SEQ ID        NO: 35, the light chain CDR 2 of SEQ ID NO: 36 and the light        chain CDR 3 of SEQ ID NO: 37;    -   (iii) in the constant domain CL of the first Fab molecule        under a) and the third Fab molecule under c) the amino acid at        position 124 is substituted by lysine (K) (numbering according        to Kabat) and the amino acid at position 123 is substituted by        lysine (K) or arginine (R), particularly by arginine (R)        (numbering according to Kabat), and wherein in the constant        domain CH1 of the first Fab molecule under a) and the third Fab        molecule under c) the amino acid at position 147 is substituted        by glutamic acid (E) (numbering according to Kabat EU index) and        the amino acid at position 213 is substituted by glutamic        acid (E) (numbering according to Kabat EU index); and    -   (iv) the first Fab molecule under a) is fused at the C-terminus        of the Fab heavy chain to the N-terminus of the Fab heavy chain        of the second Fab molecule under b), and the second Fab molecule        under b) and the third Fab molecule under c) are each fused at        the C-terminus of the Fab heavy chain to the N-terminus of one        of the subunits of the Fc domain under d).

In one embodiment, the first Fab molecule under a) and the third Fabmolecule under c) each comprise a heavy chain variable region that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:65, and a light chain variable region that is at least 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 66.

In one embodiment, the first Fab molecule under a) and the third Fabmolecule under c) each comprise the heavy chain variable region sequenceof SEQ ID NO: 65, and the light chain variable region sequence of SEQ IDNO: 66.

In one embodiment, the second Fab molecule under b) comprises a heavychain variable region that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 38, and a light chain variableregion that is at least 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 39.

In still a further embodiment, the second Fab molecule under b)comprises the heavy chain variable region sequence of SEQ ID NO: 38, andthe light chain variable region sequence of SEQ ID NO: 39.

In a particular embodiment, the bispecific antibody comprises apolypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 47, a polypeptide that is at least 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO: 148, a polypeptidethat is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 149, and a polypeptide that is at least 95%, 96%, 97%, 98%,or 99% identical to the sequence of SEQ ID NO: 150. In a furtherparticular embodiment, the bispecific antibody comprises a polypeptidesequence of SEQ ID NO: 47, a polypeptide sequence of SEQ ID NO: 148, apolypeptide sequence of SEQ ID NO: 149 and a polypeptide sequence of SEQID NO: 150.

Antibody Formats

The components of an antibody comprised in the therapeutic agent,particularly a multispecific antibody, can be fused to each other in avariety of configurations. Exemplary configurations are depicted in FIG.6 .

In particular embodiments, the antigen binding moieties comprised in theantibody are Fab molecules. In such embodiments, the first, second,third etc. antigen binding moiety may be referred to herein as first,second, third etc. Fab molecule, respectively. Furthermore, inparticular embodiments, the antibody comprises an Fc domain composed ofa first and a second subunit capable of stable association.

In some embodiments, the second Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the first or the secondsubunit of the Fc domain.

In one such embodiment, the first Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the second Fab molecule. In a specific such embodiment, theantibody essentially consists of the first and the second Fab molecule,the Fc domain composed of a first and a second subunit, and optionallyone or more peptide linkers, wherein the first Fab molecule is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the second Fab molecule, and the second Fab molecule is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the firstor the second subunit of the Fc domain. Such a configuration isschematically depicted in FIGS. 6G and 6K. Optionally, the Fab lightchain of the first Fab molecule and the Fab light chain of the secondFab molecule may additionally be fused to each other.

In another such embodiment, the first Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first orsecond subunit of the Fc domain. In a specific such embodiment, theantibody essentially consists of the first and the second Fab molecule,the Fc domain composed of a first and a second subunit, and optionallyone or more peptide linkers, wherein the first and the second Fabmolecule are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain. Such a configurationis schematically depicted in FIGS. 6A and 6D. The first and the secondFab molecule may be fused to the Fc domain directly or through a peptidelinker. In a particular embodiment the first and the second Fab moleculeare each fused to the Fc domain through an immunoglobulin hinge region.In a specific embodiment, the immunoglobulin hinge region is a humanIgG₁ hinge region, particularly where the Fc domain is an IgG₁ Fcdomain.

In other embodiments, the first Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the first or second subunitof the Fc domain.

In one such embodiment, the second Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first Fab molecule. In a specific such embodiment, theantibody essentially consists of the first and the second Fab molecule,the Fc domain composed of a first and a second subunit, and optionallyone or more peptide linkers, wherein the second Fab molecule is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first Fab molecule, and the first Fab molecule is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the first orthe second subunit of the Fc domain. Such a configuration isschematically depicted in FIGS. 6H and 6L. Optionally, the Fab lightchain of the first Fab molecule and the Fab light chain of the secondFab molecule may additionally be fused to each other.

The Fab molecules may be fused to the Fc domain or to each otherdirectly or through a peptide linker, comprising one or more aminoacids, typically about 2-20 amino acids. Peptide linkers are known inthe art and are described herein. Suitable, non-immunogenic peptidelinkers include, for example, (G₄S)_(n), (SG₄)_(n), (G₄S)_(n) orG₄(SG₄)_(n) peptide linkers. “n” is generally an integer from 1 to 10,typically from 2 to 4. In one embodiment said peptide linker has alength of at least 5 amino acids, in one embodiment a length of 5 to100, in a further embodiment of 10 to 50 amino acids. In one embodimentsaid peptide linker is (GxS)_(n) or (GxS)_(n)G_(m) with G=glycine,S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3,4 or 5 and m=0, 1, 2 or 3), in one embodiment x=4 and n=2 or 3, in afurther embodiment x=4 and n=2. In one embodiment said peptide linker is(G₄S)₂. A particularly suitable peptide linker for fusing the Fab lightchains of the first and the second Fab molecule to each other is (G₄S)₂.An exemplary peptide linker suitable for connecting the Fab heavy chainsof the first and the second Fab fragments comprises the sequence(D)-(G₄S)₂ (SEQ ID NOs 118 and 119). Another suitable such linkercomprises the sequence (G₄S)₄. Additionally, linkers may comprise (aportion of) an immunoglobulin hinge region. Particularly where a Fabmolecule is fused to the N-terminus of an Fc domain subunit, it may befused via an immunoglobulin hinge region or a portion thereof, with orwithout an additional peptide linker.

An antibody with a single antigen binding moiety (such as a Fabmolecule) capable of specific binding to a target cell antigen (forexample as shown in FIG. 6A, D, G, H, K, L) is useful, particularly incases where internalization of the target cell antigen is to be expectedfollowing binding of a high affinity antigen binding moiety. In suchcases, the presence of more than one antigen binding moiety specific forthe target cell antigen may enhance internalization of the target cellantigen, thereby reducing its availability.

In many other cases, however, it will be advantageous to have anantibody comprising two or more antigen binding moieties (such as Fabmolecules) specific for a target cell antigen (see examples shown inFIG. 6B, 6C, 6E, 6F, 6I, 6J. 6M or 6N), for example to optimizetargeting to the target site or to allow crosslinking of target cellantigens.

Accordingly, in particular embodiments, the antibody further comprises athird Fab molecule which specifically binds to the first antigen. Thefirst antigen preferably is the target cell antigen. In one embodiment,the third Fab molecule is a conventional Fab molecule. In oneembodiment, the third Fab molecule is identical to the first Fabmolecule (i.e. the first and the third Fab molecule comprise the sameheavy and light chain amino acid sequences and have the same arrangementof domains (i.e. conventional or crossover)). In a particularembodiment, the second Fab molecule specifically binds to an activatingT cell antigen, particularly CD3, and the first and third Fab moleculespecifically bind to a target cell antigen.

In alternative embodiments, the antibody further comprises a third Fabmolecule which specifically binds to the second antigen. In theseembodiments, the second antigen preferably is the target cell antigen.In one such embodiment, the third Fab molecule is a crossover Fabmolecule (a Fab molecule wherein the variable domains VH and VL or theconstant domains CL and CH1 of the Fab heavy and light chains areexchanged/replaced by each other). In one such embodiment, the third Fabmolecule is identical to the second Fab molecule (i.e. the second andthe third Fab molecule comprise the same heavy and light chain aminoacid sequences and have the same arrangement of domains (i.e.conventional or crossover)). In one such embodiment, the first Fabmolecule specifically binds to an activating T cell antigen,particularly CD3, and the second and third Fab molecule specificallybind to a target cell antigen.

In one embodiment, the third Fab molecule is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the first or second subunit ofthe Fc domain.

In a particular embodiment, the second and the third Fab molecule areeach fused at the C-terminus of the Fab heavy chain to the N-terminus ofone of the subunits of the Fc domain, and the first Fab molecule isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the second Fab molecule. In a specific suchembodiment, the antibody essentially consists of the first, the secondand the third Fab molecule, the Fc domain composed of a first and asecond subunit, and optionally one or more peptide linkers, wherein thefirst Fab molecule is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the Fab heavy chain of the second Fab molecule, andthe second Fab molecule is fused at the C-terminus of the Fab heavychain to the N-terminus of the first subunit of the Fc domain, andwherein the third Fab molecule is fused at the C-terminus of the Fabheavy chain to the N-terminus of the second subunit of the Fc domain.Such a configuration is schematically depicted in FIGS. 6B and 6E(particular embodiments, wherein the third Fab molecule is aconventional Fab molecule and preferably identical to the first Fabmolecule), and FIGS. 6I and 6M (alternative embodiments, wherein thethird Fab molecule is a crossover Fab molecule and preferably identicalto the second Fab molecule). The second and the third Fab molecule maybe fused to the Fc domain directly or through a peptide linker. In aparticular embodiment the second and the third Fab molecule are eachfused to the Fc domain through an immunoglobulin hinge region. In aspecific embodiment, the immunoglobulin hinge region is a human IgG₁hinge region, particularly where the Fc domain is an IgG₁ Fc domain.Optionally, the Fab light chain of the first Fab molecule and the Fablight chain of the second Fab molecule may additionally be fused to eachother.

In another embodiment, the first and the third Fab molecule are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain, and the second Fab molecule is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the Fabheavy chain of the first Fab molecule. In a specific such embodiment,the antibody essentially consists of the first, the second and the thirdFab molecule, the Fc domain composed of a first and a second subunit,and optionally one or more peptide linkers, wherein the second Fabmolecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first Fab molecule, and thefirst Fab molecule is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the first subunit of the Fc domain, and wherein thethird Fab molecule is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the second subunit of the Fc domain. Such aconfiguration is schematically depicted in FIGS. 6C and 6F (particularembodiments, wherein the third Fab molecule is a conventional Fabmolecule and preferably identical to the first Fab molecule) and inFIGS. 6J and 6N (alternative embodiments, wherein the third Fab moleculeis a crossover Fab molecule and preferably identical to the second Fabmolecule). The first and the third Fab molecule may be fused to the Fcdomain directly or through a peptide linker. In a particular embodimentthe first and the third Fab molecule are each fused to the Fc domainthrough an immunoglobulin hinge region. In a specific embodiment, theimmunoglobulin hinge region is a human IgG₁ hinge region, particularlywhere the Fc domain is an IgG₁ Fc domain. Optionally, the Fab lightchain of the first Fab molecule and the Fab light chain of the secondFab molecule may additionally be fused to each other.

In configurations of the antibody wherein a Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of each of thesubunits of the Fc domain through an immunoglobulin hinge regions, thetwo Fab molecules, the hinge regions and the Fc domain essentially forman immunoglobulin molecule. In a particular embodiment theimmunoglobulin molecule is an IgG class immunoglobulin. In an even moreparticular embodiment the immunoglobulin is an IgG₁ subclassimmunoglobulin. In another embodiment the immunoglobulin is an IgG4subclass immunoglobulin. In a further particular embodiment theimmunoglobulin is a human immunoglobulin. In other embodiments theimmunoglobulin is a chimeric immunoglobulin or a humanizedimmunoglobulin.

In some of the antibodies, the Fab light chain of the first Fab moleculeand the Fab light chain of the second Fab molecule are fused to eachother, optionally via a peptide linker. Depending on the configurationof the first and the second Fab molecule, the Fab light chain of thefirst Fab molecule may be fused at its C-terminus to the N-terminus ofthe Fab light chain of the second Fab molecule, or the Fab light chainof the second Fab molecule may be fused at its C-terminus to theN-terminus of the Fab light chain of the first Fab molecule. Fusion ofthe Fab light chains of the first and the second Fab molecule furtherreduces mispairing of unmatched Fab heavy and light chains, and alsoreduces the number of plasmids needed for expression of some of theantibodies.

In certain embodiments the antibody comprises a polypeptide wherein theFab light chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region), which in turn shares acarboxy-terminal peptide bond with an Fc domain subunit(VL₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)), and a polypeptide wherein the Fab heavychain of the first Fab molecule shares a carboxy-terminal peptide bondwith an Fc domain subunit (VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In someembodiments the antibody further comprises a polypeptide wherein the Fabheavy chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (VH₍₂₎-CL₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). In certainembodiments the polypeptides are covalently linked, e.g., by a disulfidebond.

In certain embodiments the antibody comprises a polypeptide wherein theFab heavy chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region), which in turn shares acarboxy-terminal peptide bond with an Fc domain subunit(VH₍₂₎-CL₍₂₎-CH2-CH3(-CH4)), and a polypeptide wherein the Fab heavychain of the first Fab molecule shares a carboxy-terminal peptide bondwith an Fc domain subunit (VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In someembodiments the antibody further comprises a polypeptide wherein the Fablight chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (VL₍₂₎-CH1₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). In certainembodiments the polypeptides are covalently linked, e.g., by a disulfidebond.

In some embodiments, the antibody comprises a polypeptide wherein theFab light chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain of the first Fabmolecule, which in turn shares a carboxy-terminal peptide bond with anFc domain subunit (VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In otherembodiments, the antibody comprises a polypeptide wherein the Fab heavychain of the first Fab molecule shares a carboxy-terminal peptide bondwith the Fab light chain variable region of the second Fab moleculewhich in turn shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the second Fab molecule (i.e. the second Fabmolecule comprises a crossover Fab heavy chain, wherein the heavy chainvariable region is replaced by a light chain variable region), which inturn shares a carboxy-terminal peptide bond with an Fc domain subunit(VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)).

In some of these embodiments the antibody further comprises a crossoverFab light chain polypeptide of the second Fab molecule, wherein the Fabheavy chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (VH₍₂₎-CL₍₂₎), and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). In others of theseembodiments the antibody further comprises a polypeptide wherein the Fabheavy chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab light chain polypeptide of the first Fabmolecule (VH₍₂₎-CL₍₂₎-VL₍₁₎-CL₍₁₎), or a polypeptide wherein the Fablight chain polypeptide of the first Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain variable regionof the second Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (VL₍₁₎-CL₍₁₎-VH₍₂₎-CL₍₂₎), as appropriate.

The antibody according to these embodiments may further comprise (i) anFc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptidewherein the Fab heavy chain of a third Fab molecule shares acarboxy-terminal peptide bond with an Fc domain subunit(VH₍₃₎-CH1₍₃₎-CH2-CH3(-CH4)) and the Fab light chain polypeptide of athird Fab molecule (VL₍₃₎-CL₍₃₎). In certain embodiments thepolypeptides are covalently linked, e.g., by a disulfide bond.

In some embodiments, the antibody comprises a polypeptide wherein theFab heavy chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain of the first Fabmolecule, which in turn shares a carboxy-terminal peptide bond with anFc domain subunit (VH₍₂₎-CL₍₂₎-VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In otherembodiments, the antibody comprises a polypeptide wherein the Fab heavychain of the first Fab molecule shares a carboxy-terminal peptide bondwith the Fab heavy chain variable region of the second Fab moleculewhich in turn shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (i.e. the second Fabmolecule comprises a crossover Fab heavy chain, wherein the heavy chainconstant region is replaced by a light chain constant region), which inturn shares a carboxy-terminal peptide bond with an Fc domain subunit(VH₍₁₎-CH1₍₁₎-VH₍₂₎-CL₍₂₎-CH2-CH3(-CH4)).

In some of these embodiments the antibody further comprises a crossoverFab light chain polypeptide of the second Fab molecule, wherein the Fablight chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (VL₍₂₎-CH1₍₂₎), and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). In others of theseembodiments the antibody further comprises a polypeptide wherein the Fablight chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab light chain polypeptide of the first Fabmolecule (VL₍₂₎-CH1₍₂₎-VL₍₁₎-CL₍₁₎), or a polypeptide wherein the Fablight chain polypeptide of the first Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain variable regionof the second Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (VL₍₁₎-CL₍₁₎-VH₍₂₎-CL₍₂₎), as appropriate.

The antibody according to these embodiments may further comprise (i) anFc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptidewherein the Fab heavy chain of a third Fab molecule shares acarboxy-terminal peptide bond with an Fc domain subunit(VH₍₃₎-CH1₍₃₎-CH2-CH3(-CH4)) and the Fab light chain polypeptide of athird Fab molecule (VL₍₃₎-CL₍₃₎). In certain embodiments thepolypeptides are covalently linked, e.g., by a disulfide bond.

In some embodiments, the first Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond Fab molecule. In certain such embodiments, the antibody does notcomprise an Fc domain. In certain embodiments, the antibody essentiallyconsists of the first and the second Fab molecule, and optionally one ormore peptide linkers, wherein the first Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the second Fab molecule. Such a configuration is schematicallydepicted in FIGS. 6O and 6S.

In other embodiments, the second Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst Fab molecule. In certain such embodiments, the antibody does notcomprise an Fc domain. In certain embodiments, the antibody essentiallyconsists of the first and the second Fab molecule, and optionally one ormore peptide linkers, wherein the second Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first Fab molecule. Such a configuration is schematicallydepicted in FIGS. 6P and 6T.

In some embodiments, the first Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond Fab molecule, and the antibody further comprises a third Fabmolecule, wherein said third Fab molecule is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst Fab molecule. In particular such embodiments, said third Fabmolecule is a conventional Fab molecule. In other such embodiments, saidthird Fab molecule is a crossover Fab molecule as described herein, i.e.a Fab molecule wherein the variable domains VH and VL or the constantdomains CL and CH1 of the Fab heavy and light chains areexchanged/replaced by each other. In certain such embodiments, theantibody essentially consists of the first, the second and the third Fabmolecule, and optionally one or more peptide linkers, wherein the firstFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second Fab molecule, and thethird Fab molecule is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the Fab heavy chain of the first Fab molecule. Such aconfiguration is schematically depicted in FIGS. 6Q and 6U (particularembodiments, wherein the third Fab molecule is a conventional Fabmolecule and preferably identical to the first Fab molecule).

In some embodiments, the first Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond Fab molecule, and the antibody further comprises a third Fabmolecule, wherein said third Fab molecule is fused at the N-terminus ofthe Fab heavy chain to the C-terminus of the Fab heavy chain of thesecond Fab molecule. In particular such embodiments, said third Fabmolecule is a crossover Fab molecule as described herein, i.e. a Fabmolecule wherein the variable domains VH and VL or the constant domainsCH1 and CL of the Fab heavy and light chains are exchanged/replaced byeach other. In other such embodiments, said third Fab molecule is aconventional Fab molecule. In certain such embodiments, the antibodyessentially consists of the first, the second and the third Fabmolecule, and optionally one or more peptide linkers, wherein the firstFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second Fab molecule, and thethird Fab molecule is fused at the N-terminus of the Fab heavy chain tothe C-terminus of the Fab heavy chain of the second Fab molecule. Such aconfiguration is schematically depicted in FIGS. 6W and 6Y (particularembodiments, wherein the third Fab molecule is a crossover Fab moleculeand preferably identical to the second Fab molecule).

In some embodiments, the second Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst Fab molecule, and the antibody further comprises a third Fabmolecule, wherein said third Fab molecule is fused at the N-terminus ofthe Fab heavy chain to the C-terminus of the Fab heavy chain of thefirst Fab molecule. In particular such embodiments, said third Fabmolecule is a conventional Fab molecule. In other such embodiments, saidthird Fab molecule is a crossover Fab molecule as described herein, i.e.a Fab molecule wherein the variable domains VH and VL or the constantdomains CH1 and CL of the Fab heavy and light chains areexchanged/replaced by each other. In certain such embodiments, theantibody essentially consists of the first, the second and the third Fabmolecule, and optionally one or more peptide linkers, wherein the secondFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first Fab molecule, and thethird Fab molecule is fused at the N-terminus of the Fab heavy chain tothe C-terminus of the Fab heavy chain of the first Fab molecule. Such aconfiguration is schematically depicted in FIGS. 6R and 6V (particularembodiments, wherein the third Fab molecule is a conventional Fabmolecule and preferably identical to the first Fab molecule).

In some embodiments, the second Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst Fab molecule, and the antibody further comprises a third Fabmolecule, wherein said third Fab molecule is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond Fab molecule. In particular such embodiments, said third Fabmolecule is a crossover Fab molecule as described herein, i.e. a Fabmolecule wherein the variable domains VH and VL or the constant domainsCH1 and CL of the Fab heavy and light chains are exchanged/replaced byeach other. In other such embodiments, said third Fab molecule is aconventional Fab molecule. In certain such embodiments, the antibodyessentially consists of the first, the second and the third Fabmolecule, and optionally one or more peptide linkers, wherein the secondFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first Fab molecule, and thethird Fab molecule is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the Fab heavy chain of the second Fab molecule. Such aconfiguration is schematically depicted in FIGS. 6X and 6Z (particularembodiments, wherein the third Fab molecule is a crossover Fab moleculeand preferably identical to the first Fab molecule).

In certain embodiments the antibody comprises a polypeptide wherein theFab heavy chain of the first Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain variable region of the second Fabmolecule, which in turn shares a carboxy-terminal peptide bond with theFab heavy chain constant region of the second Fab molecule (i.e. thesecond Fab molecule comprises a crossover Fab heavy chain, wherein theheavy chain variable region is replaced by a light chain variableregion) (VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎). In some embodiments the antibodyfurther comprises a polypeptide wherein the Fab heavy chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab light chain constant region of the second Fab molecule(VH₍₂₎-CL₍₂₎) and the Fab light chain polypeptide of the first Fabmolecule (VL₍₁₎-CL₍₁₎).

In certain embodiments the antibody comprises a polypeptide wherein theFab light chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain of the first Fabmolecule (VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎). In some embodiments the antibodyfurther comprises a polypeptide wherein the Fab heavy chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab light chain constant region of the second Fab molecule(VH₍₂₎-CL₍₂₎) and the Fab light chain polypeptide of the first Fabmolecule (VL₍₁₎-CL₍₁₎).

In certain embodiments the antibody comprises a polypeptide wherein theFab heavy chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain of the first Fabmolecule (VH₍₂₎-CL₍₂₎-VH₍₁₎-CH1₍₁₎). In some embodiments the antibodyfurther comprises a polypeptide wherein the Fab light chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab heavy chain constant region of the second Fab molecule(VL₍₂₎-CH1₍₂₎) and the Fab light chain polypeptide of the first Fabmolecule (VL₍₁₎-CL₍₁₎).

In certain embodiments the antibody comprises a polypeptide wherein theFab heavy chain of a third Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain of the first Fab molecule, whichin turn shares a carboxy-terminal peptide bond with the Fab light chainvariable region of the second Fab molecule, which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region)(VH₍₃₎-CH1₍₃₎-VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎). In some embodiments theantibody further comprises a polypeptide wherein the Fab heavy chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (VH₍₂₎-CL₍₂₎) and the Fab light chain polypeptide of the firstFab molecule (VL₍₁₎-CL₍₁₎). In some embodiments the antibody furthercomprises the Fab light chain polypeptide of a third Fab molecule(VL₍₃₎-CL₍₃₎).

In certain embodiments the antibody comprises a polypeptide wherein theFab heavy chain of a third Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain of the first Fab molecule, whichin turn shares a carboxy-terminal peptide bond with the Fab heavy chainvariable region of the second Fab molecule, which in turn shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region)(VH₍₃₎-CH1₍₃₎-VH₍₁₎-CH1₍₁₎-VH₍₂₎-CL₍₂₎). In some embodiments theantibody further comprises a polypeptide wherein the Fab light chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the second Fabmolecule (VL₍₂₎-CH1₍₂₎) and the Fab light chain polypeptide of the firstFab molecule (VL₍₁₎-CL₍₁₎). In some embodiments the antibody furthercomprises the Fab light chain polypeptide of a third Fab molecule(VL₍₃₎-CL₍₃₎).

In certain embodiments the antibody comprises a polypeptide wherein theFab light chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain of the first Fabmolecule, which in turn shares a carboxy-terminal peptide bond with theFab heavy chain of a third Fab molecule(VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎-VH₍₃₎-CH1₍₃₎). In some embodiments theantibody further comprises a polypeptide wherein the Fab heavy chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (VH₍₂₎-CL₍₂₎) and the Fab light chain polypeptide of the firstFab molecule (VL₍₁₎-CL₍₁₎). In some embodiments the antibody furthercomprises the Fab light chain polypeptide of a third Fab molecule(VL₍₃₎-CL₍₃₎).

In certain embodiments the antibody comprises a polypeptide wherein theFab heavy chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain of the first Fabmolecule, which in turn shares a carboxy-terminal peptide bond with theFab heavy chain of a third Fab molecule(VH₍₂₎-CL₍₂₎-VH₍₁₎-CH1₍₁₎-VH₍₃₎-CH1₍₃₎). In some embodiments theantibody further comprises a polypeptide wherein the Fab light chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the second Fabmolecule (VL₍₂₎-CH1₍₂₎) and the Fab light chain polypeptide of the firstFab molecule (VL₍₁₎-CL₍₁₎). In some embodiments the antibody furthercomprises the Fab light chain polypeptide of a third Fab molecule(VL₍₃₎-CL₍₃₎).

In certain embodiments the antibody comprises a polypeptide wherein theFab heavy chain of the first Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain variable region of the second Fabmolecule, which in turn shares a carboxy-terminal peptide bond with theFab heavy chain constant region of the second Fab molecule (i.e. thesecond Fab molecule comprises a crossover Fab heavy chain, wherein theheavy chain variable region is replaced by a light chain variableregion), which in turn shares a carboxy-terminal peptide bond with theFab light chain variable region of a third Fab molecule, which in turnshares a carboxy-terminal peptide bond with the Fab heavy chain constantregion of a third Fab molecule (i.e. the third Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region)(VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎-VL₍₃₎-CH1₍₃₎). In some embodiments theantibody further comprises a polypeptide wherein the Fab heavy chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (VH₍₂₎-CL₍₂₎) and the Fab light chain polypeptide of the firstFab molecule (VL₍₁₎-CL₍₁₎). In some embodiments the antibody furthercomprises a polypeptide wherein the Fab heavy chain variable region of athird Fab molecule shares a carboxy-terminal peptide bond with the Fablight chain constant region of a third Fab molecule (VH₍₃₎-CL₍₃₎).

In certain embodiments the antibody comprises a polypeptide wherein theFab heavy chain of the first Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain variable region of the second Fabmolecule, which in turn shares a carboxy-terminal peptide bond with theFab light chain constant region of the second Fab molecule (i.e. thesecond Fab molecule comprises a crossover Fab heavy chain, wherein theheavy chain constant region is replaced by a light chain constantregion), which in turn shares a carboxy-terminal peptide bond with theFab heavy chain variable region of a third Fab molecule, which in turnshares a carboxy-terminal peptide bond with the Fab light chain constantregion of a third Fab molecule (i.e. the third Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region)(VH₍₁₎-CH1₍₁₎-VH₍₂₎-CL₍₂₎-VH₍₃₎-CL₍₃₎). In some embodiments the antibodyfurther comprises a polypeptide wherein the Fab light chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab heavy chain constant region of the second Fab molecule(VL₍₂₎-CH1₍₂₎) and the Fab light chain polypeptide of the first Fabmolecule (VL₍₁₎-CL₍₁₎). In some embodiments the antibody furthercomprises a polypeptide wherein the Fab light chain variable region of athird Fab molecule shares a carboxy-terminal peptide bond with the Fabheavy chain constant region of a third Fab molecule (VL₍₃₎-CH1₍₃₎).

In certain embodiments the antibody comprises a polypeptide wherein theFab light chain variable region of a third Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof a third Fab molecule (i.e. the third Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region), which in turn shares acarboxy-terminal peptide bond with the Fab light chain variable regionof the second Fab molecule, which in turn shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region), which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain of the first Fab molecule(VL₍₃₎-CH1₍₃₎-VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎). In some embodiments theantibody further comprises a polypeptide wherein the Fab heavy chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (VH₍₂₎-CL₍₂₎) and the Fab light chain polypeptide of the firstFab molecule (VL₍₁₎-CL₍₁₎). In some embodiments the antibody furthercomprises a polypeptide wherein the Fab heavy chain variable region of athird Fab molecule shares a carboxy-terminal peptide bond with the Fablight chain constant region of a third Fab molecule (VH₍₃₎-CL₍₃₎).

In certain embodiments the antibody comprises a polypeptide wherein theFab heavy chain variable region of a third Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof a third Fab molecule (i.e. the third Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain variable regionof the second Fab molecule, which in turn shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain constant region is replaced by a lightchain constant region), which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain of the first Fab molecule(VH₍₃₎-CL₍₃₎-VH₍₂₎-CL₍₂₎-VH₍₁₎-CH1₍₁₎). In some embodiments the antibodyfurther comprises a polypeptide wherein the Fab light chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab heavy chain constant region of the second Fab molecule(VL₍₂₎-CH1₍₂₎) and the Fab light chain polypeptide of the first Fabmolecule (VL₍₁₎-CL₍₁₎). In some embodiments the antibody furthercomprises a polypeptide wherein the Fab light chain variable region of athird Fab molecule shares a carboxy-terminal peptide bond with the Fabheavy chain constant region of a third Fab molecule (VL₍₃₎-CH1₍₃₎).

According to any of the above embodiments, components of the antibody(e.g. Fab molecules, Fc domain) may be fused directly or through variouslinkers, particularly peptide linkers comprising one or more aminoacids, typically about 2-20 amino acids, that are described herein orare known in the art. Suitable, non-immunogenic peptide linkers include,for example, (G₄S)_(n), (SG₄)_(n), (G₄S)_(n) or G₄(SG₄)_(n) peptidelinkers, wherein n is generally an integer from 1 to 10, typically from2 to 4.

Fc Domain

An antibody, e.g. a bispecific antibody or an immunoconjugate, comprisedin the therapeutic agent may comprise an Fc domain which consists of apair of polypeptide chains comprising heavy chain domains of an antibodymolecule. For example, the Fc domain of an immunoglobulin G (IgG)molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgGheavy chain constant domains. The two subunits of the Fc domain arecapable of stable association with each other.

In one embodiment, the Fc domain is an IgG Fc domain. In a particularembodiment the Fc domain is an IgG₁ Fc domain. In another embodiment theFc domain is an IgG₄ Fc domain. In a more specific embodiment, the Fcdomain is an IgG₄ Fc domain comprising an amino acid substitution atposition S228 (Kabat numbering), particularly the amino acidsubstitution S228P. This amino acid substitution reduces in vivo Fab armexchange of IgG₄ antibodies (see Stubenrauch et al., Drug Metabolism andDisposition 38, 84-91 (2010)). In a further particular embodiment the Fcdomain is human. An exemplary sequence of a human IgG₁ Fc region isgiven in SEQ ID NO: 30.

(i) Fc Domain Modifications Promoting Heterodimerization

Antibodies, particularly bispecific antibodies or immunoconjugates,comprised in the therapeutic agent may comprise different components(e.g. antigen binding domains, cytokines) fused to one or the other ofthe two subunits of the Fc domain, thus the two subunits of the Fcdomain are typically comprised in two non-identical polypeptide chains.Recombinant co-expression of these polypeptides and subsequentdimerization leads to several possible combinations of the twopolypeptides. To improve the yield and purity of such antibodies inrecombinant production, it will thus be advantageous to introduce in theFc domain of the antibody a modification promoting the association ofthe desired polypeptides.

Accordingly, in particular embodiments the Fc domain comprises amodification promoting the association of the first and the secondsubunit of the Fc domain. The site of most extensive protein-proteininteraction between the two subunits of a human IgG Fc domain is in theCH3 domain of the Fc domain. Thus, in one embodiment said modificationis in the CH3 domain of the Fc domain.

There exist several approaches for modifications in the CH3 domain ofthe Fc domain in order to enforce heterodimerization, which are welldescribed e.g. in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205,WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, inall such approaches the CH3 domain of the first subunit of the Fc domainand the CH3 domain of the second subunit of the Fc domain are bothengineered in a complementary manner so that each CH3 domain (or theheavy chain comprising it) can no longer homodimerize with itself but isforced to heterodimerize with the complementarily engineered other CH3domain (so that the first and second CH3 domain heterodimerize and nohomodimers between the two first or the two second CH3 domains areformed). These different approaches for improved heavy chainheterodimerization are contemplated as different alternatives incombination with heavy-light chain modifications (e.g. variable orconstant region exchange/replacement in Fab arms, or introduction ofsubstitutions of charged amino acids with opposite charges in the CH1/CLinterface) which reduce light chain mispairing and Bence Jones-type sideproducts.

In a specific embodiment said modification promoting the association ofthe first and the second subunit of the Fc domain is a so-called“knob-into-hole” modification, comprising a “knob” modification in oneof the two subunits of the Fc domain and a “hole” modification in theother one of the two subunits of the Fc domain.

The knob-into-hole technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine).

Accordingly, in a particular embodiment, in the CH3 domain of the firstsubunit of the Fc domain an amino acid residue is replaced with an aminoacid residue having a larger side chain volume, thereby generating aprotuberance within the CH3 domain of the first subunit which ispositionable in a cavity within the CH3 domain of the second subunit,and in the CH3 domain of the second subunit of the Fc domain an aminoacid residue is replaced with an amino acid residue having a smallerside chain volume, thereby generating a cavity within the CH3 domain ofthe second subunit within which the protuberance within the CH3 domainof the first subunit is positionable.

Preferably said amino acid residue having a larger side chain volume isselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), and tryptophan (W).

Preferably said amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), and valine (V).

The protuberance and cavity can be made by altering the nucleic acidencoding the polypeptides, e.g. by site-specific mutagenesis, or bypeptide synthesis.

In a specific embodiment, in the CH3 domain of the first subunit of theFc domain (the “knobs” subunit) the threonine residue at position 366 isreplaced with a tryptophan residue (T366W), and in the CH3 domain of thesecond subunit of the Fc domain (the “hole” subunit) the tyrosineresidue at position 407 is replaced with a valine residue (Y407V). Inone embodiment, in the second subunit of the Fc domain additionally thethreonine residue at position 366 is replaced with a serine residue(T366S) and the leucine residue at position 368 is replaced with analanine residue (L368A) (numberings according to Kabat EU index).

In yet a further embodiment, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C) or the glutamic acid residue at position 356 isreplaced with a cysteine residue (E356C), and in the second subunit ofthe Fc domain additionally the tyrosine residue at position 349 isreplaced by a cysteine residue (Y349C) (numberings according to Kabat EUindex). Introduction of these two cysteine residues results in formationof a disulfide bridge between the two subunits of the Fc domain, furtherstabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

In a particular embodiment, the first subunit of the Fc domain comprisesamino acid substitutions S354C and T366W, and the second subunit of theFc domain comprises amino acid substitutions Y349C, T366S, L368A andY407V (numbering according to Kabat EU index).

In a particular embodiment the mutant IL-2 polypeptide in theimmunoconjugate described herein, or the CD3 antigen binding moiety inthe bispecific antibody described herein, is fused to the first subunitof the Fc domain (comprising the “knob” modification). Without wishingto be bound by theory, fusion of the IL-2 polypeptide or CD3 antigenbinding moiety to the knob-containing subunit of the Fc domain will(further) minimize the generation of immunoconjugates comprising twoIL-2 polypeptides or bispecific antibodies comprising two CD3 antigenbinding moieties, respectively (steric clash of two knob-containingpolypeptides).

Other techniques of CH3-modification for enforcing theheterodimerization are contemplated as alternatives according to theinvention and are described e.g. in WO 96/27011, WO 98/050431, EP1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304,WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO2013/096291.

In one embodiment the heterodimerization approach described in EP1870459 A1, is used alternatively. This approach is based on theintroduction of charged amino acids with opposite charges at specificamino acid positions in the CH3/CH3 domain interface between the twosubunits of the Fc domain. One preferred embodiment are amino acidmutations R409D; K370E in one of the two CH3 domains (of the Fc domain)and amino acid mutations D399K; E357K in the other one of the CH3domains of the Fc domain (numbering according to Kabat EU index).

In another embodiment the antibody comprises amino acid mutation T366Win the CH3 domain of the first subunit of the Fc domain and amino acidmutations T366S, L368A, Y407V in the CH3 domain of the second subunit ofthe Fc domain, and additionally amino acid mutations R409D; K370E in theCH3 domain of the first subunit of the Fc domain and amino acidmutations D399K; E357K in the CH3 domain of the second subunit of the Fcdomain (numberings according to Kabat EU index).

In another embodiment the antibody comprises amino acid mutations S354C,T366W in the CH3 domain of the first subunit of the Fc domain and aminoacid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of thesecond subunit of the Fc domain, or the antibody comprises amino acidmutations Y349C, T366W in the CH3 domain of the first subunit of the Fcdomain and amino acid mutations S354C, T366S, L368A, Y407V in the CH3domains of the second subunit of the Fc domain and additionally aminoacid mutations R409D; K370E in the CH3 domain of the first subunit ofthe Fc domain and amino acid mutations D399K; E357K in the CH3 domain ofthe second subunit of the Fc domain (all numberings according to KabatEU index).

In one embodiment the heterodimerization approach described in WO2013/157953 is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutation T366K and a second CH3 domain comprisesamino acid mutation L351D (numberings according to Kabat EU index). In afurther embodiment the first CH3 domain comprises further amino acidmutation L351K. In a further embodiment the second CH3 domain comprisesfurther an amino acid mutation selected from Y349E, Y349D and L368E(preferably L368E) (numberings according to Kabat EU index).

In one embodiment the heterodimerization approach described in WO2012/058768 is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutations L351Y, Y407A and a second CH3 domaincomprises amino acid mutations T366A, K409F. In a further embodiment thesecond CH3 domain comprises a further amino acid mutation at positionT411, D399, 5400, F405, N390, or K392, e.g. selected from a) T411N,T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y orD399K, c) S400E, S400D, S400R, or S400K, d) F4051, F405M, F405T, F405S,F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L,K392F or K392E (numberings according to Kabat EU index). In a furtherembodiment a first CH3 domain comprises amino acid mutations L351Y,Y407A and a second CH3 domain comprises amino acid mutations T366V,K409F. In a further embodiment a first CH3 domain comprises amino acidmutation Y407A and a second CH3 domain comprises amino acid mutationsT366A, K409F. In a further embodiment the second CH3 domain furthercomprises amino acid mutations K392E, T411E, D399R and S400R (numberingsaccording to Kabat EU index).

In one embodiment the heterodimerization approach described in WO2011/143545 is used alternatively, e.g. with the amino acid modificationat a position selected from the group consisting of 368 and 409(numbering according to Kabat EU index).

In one embodiment the heterodimerization approach described in WO2011/090762, which also uses the knobs-into-holes technology describedabove, is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutation T366W and a second CH3 domain comprisesamino acid mutation Y407A. In one embodiment a first CH3 domaincomprises amino acid mutation T366Y and a second CH3 domain comprisesamino acid mutation Y407T (numberings according to Kabat EU index).

In one embodiment the antibody or its Fc domain is of IgG2 subclass andthe heterodimerization approach described in WO 2010/129304 is usedalternatively.

In an alternative embodiment a modification promoting association of thefirst and the second subunit of the Fc domain comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the two Fc domainsubunits by charged amino acid residues so that homodimer formationbecomes electrostatically unfavorable but heterodimerizationelectrostatically favorable. In one such embodiment a first CH3 domaincomprises amino acid substitution of K392 or N392 with a negativelycharged amino acid (e.g. glutamic acid (E), or aspartic acid (D),preferably K392D or N392D) and a second CH3 domain comprises amino acidsubstitution of D399, E356, D356, or E357 with a positively chargedamino acid (e.g. lysine (K) or arginine (R), preferably D399K, E356K,D356K, or E357K, and more preferably D399K and E356K). In a furtherembodiment the first CH3 domain further comprises amino acidsubstitution of K409 or R409 with a negatively charged amino acid (e.g.glutamic acid (E), or aspartic acid (D), preferably K409D or R409D). Ina further embodiment the first CH3 domain further or alternativelycomprises amino acid substitution of K439 and/or K370 with a negativelycharged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (allnumberings according to Kabat EU index).

In yet a further embodiment the heterodimerization approach described inWO 2007/147901 is used alternatively. In one embodiment a first CH3domain comprises amino acid mutations K253E, D282K, and K322D and asecond CH3 domain comprises amino acid mutations D239K, E240K, and K292D(numberings according to Kabat EU index).

In still another embodiment the heterodimerization approach described inWO 2007/110205 can be used alternatively.

In one embodiment, the first subunit of the Fc domain comprises aminoacid substitutions K392D and K409D, and the second subunit of the Fcdomain comprises amino acid substitutions D356K and D399K (numberingaccording to Kabat EU index).

(ii) Fc Domain Modifications Reducing Fc Receptor Binding and/orEffector Function

The Fc domain confers to an antibody, such as a bispecific antibody orimmunoconjugate, favorable pharmacokinetic properties, including a longserum half-life which contributes to good accumulation in the targettissue and a favorable tissue-blood distribution ratio. At the same timeit may, however, lead to undesirable targeting of the antibody to cellsexpressing Fc receptors rather than to the preferred antigen-bearingcells. Moreover, the co-activation of Fc receptor signaling pathways maylead to cytokine release which, in combination with otherimmunostimulatory properties the antibody may have and the longhalf-life of the antibody, results in excessive activation of cytokinereceptors and severe side effects upon systemic administration.

Accordingly, in particular embodiments, the Fc domain of the antibody,particularly bispecific antibody or immunoconjugate, comprised in thetherapeutic agent exhibits reduced binding affinity to an Fc receptorand/or reduced effector function, as compared to a native IgG₁ Fcdomain. In one such embodiment the Fc domain (or the molecule, e.g.antibody, comprising said Fc domain) exhibits less than 50%, preferablyless than 20%, more preferably less than 10% and most preferably lessthan 5% of the binding affinity to an Fc receptor, as compared to anative IgG₁ Fc domain (or a corresponding molecule comprising a nativeIgG₁ Fc domain), and/or less than 50%, preferably less than 20%, morepreferably less than 10% and most preferably less than 5% of theeffector function, as compared to a native IgG₁ Fc domain (or acorresponding molecule comprising a native IgG₁ Fc domain). In oneembodiment, the Fc domain (or the molecule, e.g. antibody, comprisingsaid Fc domain) does not substantially bind to an Fc receptor and/orinduce effector function. In a particular embodiment the Fc receptor isan Fcγ receptor. In one embodiment the Fc receptor is a human Fcreceptor. In one embodiment the Fc receptor is an activating Fcreceptor. In a specific embodiment the Fc receptor is an activatinghuman Fey receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa,most specifically human FcγRIIIa. In one embodiment the effectorfunction is one or more selected from the group of CDC, ADCC, ADCP, andcytokine secretion. In a particular embodiment the effector function isADCC. In one embodiment the Fc domain exhibits substantially similarbinding affinity to neonatal Fc receptor (FcRn), as compared to a nativeIgG₁ Fc domain. Substantially similar binding to FcRn is achieved whenthe Fc domain (or the molecule, e.g. antibody, comprising said Fcdomain) exhibits greater than about 70%, particularly greater than about80%, more particularly greater than about 90% of the binding affinity ofa native IgG₁ Fc domain (or the corresponding molecule comprising anative IgG₁ Fc domain) to FcRn.

In certain embodiments the Fc domain is engineered to have reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a non-engineered Fc domain. In particular embodiments, theFc domain comprises one or more amino acid mutation that reduces thebinding affinity of the Fc domain to an Fc receptor and/or effectorfunction. Typically, the same one or more amino acid mutation is presentin each of the two subunits of the Fc domain. In one embodiment theamino acid mutation reduces the binding affinity of the Fc domain to anFc receptor. In one embodiment the amino acid mutation reduces thebinding affinity of the Fc domain to an Fc receptor by at least 2-fold,at least 5-fold, or at least 10-fold. In embodiments where there is morethan one amino acid mutation that reduces the binding affinity of the Fcdomain to the Fc receptor, the combination of these amino acid mutationsmay reduce the binding affinity of the Fc domain to an Fc receptor by atleast 10-fold, at least 20-fold, or even at least 50-fold. In oneembodiment the molecule, e.g. antibody, comprising an engineered Fcdomain exhibits less than 20%, particularly less than 10%, moreparticularly less than 5% of the binding affinity to an Fc receptor ascompared to a corresponding molecule comprising a non-engineered Fcdomain. In a particular embodiment the Fc receptor is an Fcγ receptor.In some embodiments the Fc receptor is a human Fc receptor. In someembodiments the Fc receptor is an activating Fc receptor. In a specificembodiment the Fc receptor is an activating human Fcγ receptor, morespecifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically humanFcγRIIIa. Preferably, binding to each of these receptors is reduced. Insome embodiments binding affinity to a complement component,specifically binding affinity to C1q, is also reduced. In one embodimentbinding affinity to neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn, i.e. preservation of the bindingaffinity of the Fc domain to said receptor, is achieved when the Fcdomain (or the molecule, e.g. antibody, comprising said Fc domain)exhibits greater than about 70% of the binding affinity of anon-engineered form of the Fc domain (or a corresponding moleculecomprising said non-engineered form of the Fc domain) to FcRn. The Fcdomain, or molecule (e.g. antibody) comprising said Fc domain, mayexhibit greater than about 80% and even greater than about 90% of suchaffinity. In certain embodiments the Fc domain is engineered to havereduced effector function, as compared to a non-engineered Fc domain.The reduced effector function can include, but is not limited to, one ormore of the following: reduced complement dependent cytotoxicity (CDC),reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reducedantibody-dependent cellular phagocytosis (ADCP), reduced cytokinesecretion, reduced immune complex-mediated antigen uptake byantigen-presenting cells, reduced binding to NK cells, reduced bindingto macrophages, reduced binding to monocytes, reduced binding topolymorphonuclear cells, reduced direct signaling inducing apoptosis,reduced crosslinking of target-bound antibodies, reduced dendritic cellmaturation, or reduced T cell priming. In one embodiment the reducedeffector function is one or more selected from the group of reduced CDC,reduced ADCC, reduced ADCP, and reduced cytokine secretion. In aparticular embodiment the reduced effector function is reduced ADCC. Inone embodiment the reduced ADCC is less than 20% of the ADCC induced bya non-engineered Fc domain (or a corresponding molecule comprising anon-engineered Fc domain).

In one embodiment the amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function isan amino acid substitution. In one embodiment the Fc domain comprises anamino acid substitution at a position selected from the group of E233,L234, L235, N297, P331 and P329 (numberings according to Kabat EUindex). In a more specific embodiment the Fc domain comprises an aminoacid substitution at a position selected from the group of L234, L235and P329 (numberings according to Kabat EU index). In some embodimentsthe Fc domain comprises the amino acid substitutions L234A and L235A(numberings according to Kabat EU index). In one such embodiment, the Fcdomain is an IgG₁ Fc domain, particularly a human IgG₁ Fc domain. In oneembodiment the Fc domain comprises an amino acid substitution atposition P329. In a more specific embodiment the amino acid substitutionis P329A or P329G, particularly P329G (numberings according to Kabat EUindex). In one embodiment the Fc domain comprises an amino acidsubstitution at position P329 and a further amino acid substitution at aposition selected from E233, L234, L235, N297 and P331 (numberingsaccording to Kabat EU index). In a more specific embodiment the furtheramino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D orP331S. In particular embodiments the Fc domain comprises amino acidsubstitutions at positions P329, L234 and L235 (numberings according toKabat EU index). In more particular embodiments the Fc domain comprisesthe amino acid mutations L234A, L235A and P329G (“P329G LALA”). In onesuch embodiment, the Fc domain is an IgG₁ Fc domain, particularly ahuman IgG₁ Fc domain. The “P329G LALA” combination of amino acidsubstitutions almost completely abolishes Fcγ receptor (as well ascomplement) binding of a human IgG₁ Fc domain, as described in PCTpublication no. WO 2012/130831, incorporated herein by reference in itsentirety. WO 2012/130831 also describes methods of preparing such mutantFc domains and methods for determining its properties such as Fcreceptor binding or effector functions.

IgG₄ antibodies exhibit reduced binding affinity to Fc receptors andreduced effector functions as compared to IgG₁ antibodies. Hence, insome embodiments the Fc domain is an IgG₄ Fc domain, particularly ahuman IgG₄ Fc domain. In one embodiment the IgG₄ Fc domain comprisesamino acid substitutions at position S228, specifically the amino acidsubstitution S228P (numberings according to Kabat EU index). To furtherreduce its binding affinity to an Fc receptor and/or its effectorfunction, in one embodiment the IgG4 Fc domain comprises an amino acidsubstitution at position L235, specifically the amino acid substitutionL235E (numberings according to Kabat EU index). In another embodiment,the IgG4 Fc domain comprises an amino acid substitution at positionP329, specifically the amino acid substitution P329G (numberingsaccording to Kabat EU index). In a particular embodiment, the IgG₄ Fcdomain comprises amino acid substitutions at positions S228, L235 andP329, specifically amino acid substitutions S228P, L235E and P329G(numberings according to Kabat EU index). Such IgG₄ Fc domain mutantsand their Fcγ receptor binding properties are described in PCTpublication no. WO 2012/130831, incorporated herein by reference in itsentirety.

In a particular embodiment the Fc domain exhibiting reduced bindingaffinity to an Fc receptor and/or reduced effector function, as comparedto a native IgG₁ Fc domain, is a human IgG₁ Fc domain comprising theamino acid substitutions L234A, L235A and optionally P329G, or a humanIgG4 Fc domain comprising the amino acid substitutions S228P, L235E andoptionally P329G (numberings according to Kabat EU index).

In certain embodiments N-glycosylation of the Fc domain has beeneliminated. In one such embodiment the Fc domain comprises an amino acidmutation at position N297, particularly an amino acid substitutionreplacing asparagine by alanine (N297A) or aspartic acid (N297D) orglycine (N297G) (numberings according to Kabat EU index).

In addition to the Fc domains described hereinabove and in PCTpublication no. WO 2012/130831, Fc domains with reduced Fc receptorbinding and/or effector function also include those with substitution ofone or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329(U.S. Pat. No. 6,737,056) (numberings according to Kabat EU index). SuchFc mutants include Fc mutants with substitutions at two or more of aminoacid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine(U.S. Pat. No. 7,332,581).

Mutant Fc domains can be prepared by amino acid deletion, substitution,insertion or modification using genetic or chemical methods well knownin the art. Genetic methods may include site-specific mutagenesis of theencoding DNA sequence, PCR, gene synthesis, and the like. The correctnucleotide changes can be verified for example by sequencing.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. Alternatively, binding affinity ofFc domains or molecules comprising an Fc domain for Fc receptors may beevaluated using cell lines known to express particular Fc receptors,such as human NK cells expressing FcγIIIa receptor.

Effector function of an Fc domain, or a molecule (e.g. an antibody)comprising an Fc domain, can be measured by methods known in the art. Asuitable assay for measuring ADCC is described herein. Other examples ofin vitro assays to assess ADCC activity of a molecule of interest aredescribed in U.S. Pat. No. 5,500,362; Hellstrom et al. Proc Natl AcadSci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad SciUSA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., JExp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assaysmethods may be employed (see, for example, ACTI™ non-radioactivecytotoxicity assay for flow cytometry (CellTechnology, Inc. MountainView, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega,Madison, WI)). Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g. in a animal model such as thatdisclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

In some embodiments, binding of the Fc domain to a complement component,specifically to C1q, is reduced. Accordingly, in some embodimentswherein the Fc domain is engineered to have reduced effector function,said reduced effector function includes reduced CDC. C1q binding assaysmay be carried out to determine whether the Fc domain, or molecule (e.g.antibody) comprising the Fc domain, is able to bind C1q and hence hasCDC activity. See e.g., C1q and C3c binding ELISA in WO 2006/029879 andWO 2005/100402. To assess complement activation, a CDC assay may beperformed (see, for example, Gazzano-Santoro et al., J Immunol Methods202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Craggand Glennie, Blood 103, 2738-2743 (2004)).

Antigen Binding Moieties

The antibody comprised in the therapeutic agent may be bispecific, i.e.it comprises at least two antigen binding moieties capable of specificbinding to two distinct antigenic determinants. According to particularembodiments, the antigen binding moieties are Fab molecules (i.e.antigen binding domains composed of a heavy and a light chain, eachcomprising a variable and a constant domain). In one embodiment said Fabmolecules are human. In another embodiment said Fab molecules arehumanized. In yet another embodiment said Fab molecules comprise humanheavy and light chain constant domains.

In some embodiments, at least one of the antigen binding moieties is acrossover Fab molecule. Such modification reduces mispairing of heavyand light chains from different Fab molecules, thereby improving theyield and purity of the antibody in recombinant production. In aparticular crossover Fab molecule useful for the antibody, the variabledomains of the Fab light chain and the Fab heavy chain (VL and VH,respectively) are exchanged. Even with this domain exchange, however,the preparation of the antibody may comprise certain side products dueto a so-called Bence Jones-type interaction between mispaired heavy andlight chains (see Schaefer et al, PNAS, 108 (2011) 11187-11191). Tofurther reduce mispairing of heavy and light chains from different Fabmolecules and thus increase the purity and yield of the desiredantibody, charged amino acids with opposite charges may be introduced atspecific amino acid positions in the CH1 and CL domains of either theFab molecule(s) specifically binding to a target cell antigen, or theFab molecule specifically binding to an activating T cell antigen.Charge modifications are made either in the conventional Fab molecule(s)comprised in the antibody (such as shown e.g. in FIGS. 6A-C, G-J), or inthe VH/VL crossover Fab molecule(s) comprised in the antibody (such asshown e.g. in FIG. 6D-F, K-N) (but not in both). In particularembodiments, the charge modifications are made in the conventional Fabmolecule(s) comprised in the antibody (which in particular embodimentsspecifically bind(s) to the target cell antigen).

In a particular embodiment according to the invention, the antibody iscapable of simultaneous binding to a target cell antigen, particularly atumor cell antigen, and an activating T cell antigen, particularly CD3.In one embodiment, the antibody is capable of crosslinking a T cell anda target cell by simultaneous binding to a target cell antigen and anactivating T cell antigen. In an even more particular embodiment, suchsimultaneous binding results in lysis of the target cell, particularly atumor cell. In one embodiment, such simultaneous binding results inactivation of the T cell. In other embodiments, such simultaneousbinding results in a cellular response of a T lymphocyte, particularly acytotoxic T lymphocyte, selected from the group of: proliferation,differentiation, cytokine secretion, cytotoxic effector moleculerelease, cytotoxic activity, and expression of activation markers. Inone embodiment, binding of the antibody to the activating T cellantigen, particularly CD3, without simultaneous binding to the targetcell antigen does not result in T cell activation.

In one embodiment, the antibody is capable of re-directing cytotoxicactivity of a T cell to a target cell. In a particular embodiment, saidre-direction is independent of MHC-mediated peptide antigen presentationby the target cell and and/or specificity of the T cell.

Particularly, a T cell according to any of the embodiments of theinvention is a cytotoxic T cell. In some embodiments the T cell is aCD4⁺ or a CD8⁺ T cell, particularly a CD8⁺ T cell.

(i) Activating T Cell Antigen Binding Moiety

In some embodiments, an antibody comprised in the therapeutic agent,particularly a bispecific antibody, comprises at least one antigenbinding moiety, particularly a Fab molecule, which specifically binds toan activating T cell antigen (also referred to herein as an “activatingT cell antigen binding moiety, or activating T cell antigen binding Fabmolecule”). In a particular embodiment, the antibody comprises not morethan one antigen binding moiety capable of specific binding to anactivating T cell antigen. In one embodiment, the antibody providesmonovalent binding to the activating T cell antigen.

In particular embodiments, the antigen binding moiety which specificallybinds an activating T cell antigen is a crossover Fab molecule asdescribed herein, i.e. a Fab molecule wherein the variable domains VHand VL or the constant domains CH1 and CL of the Fab heavy and lightchains are exchanged/replaced by each other. In such embodiments, theantigen binding moiety(ies) which specifically binds a target cellantigen is preferably a conventional Fab molecule. In embodiments wherethere is more than one antigen binding moiety, particularly Fabmolecule, which specifically binds to a target cell antigen comprised inthe antibody, the antigen binding moiety which specifically binds to anactivating T cell antigen preferably is a crossover Fab molecule and theantigen binding moieties which specifically bind to a target cellantigen are conventional Fab molecules.

In alternative embodiments, the antigen binding moiety whichspecifically binds an activating T cell antigen is a conventional Fabmolecule. In such embodiments, the antigen binding moiety(ies) whichspecifically binds a target cell antigen is a crossover Fab molecule asdescribed herein, i.e. a Fab molecule wherein the variable domains VHand VL or the constant domains CH1 and CL of the Fab heavy and lightchains are exchanged/replaced by each other.

In one embodiment, the activating T cell antigen is selected from thegroup consisting of CD3, CD28, CD137 (also known as 4-1BB), CD40, CD226,OX40, GITR, CD27, HVEM, and CD127.

In a particular embodiment, the activating T cell antigen is CD3,particularly human CD3 (SEQ ID NO: 115) or cynomolgus CD3 (SEQ ID NO:116), most particularly human CD3. In a particular embodiment theactivating T cell antigen binding moiety is cross-reactive for (i.e.specifically binds to) human and cynomolgus CD3. In some embodiments,the activating T cell antigen is the epsilon subunit of CD3 (CD3epsilon).

In some embodiments, the activating T cell antigen binding moietyspecifically binds to CD3, particularly CD3 epsilon, and comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO:34 and at least one light chain CDR selected from the group of SEQ IDNO: 35, SEQ ID NO: 36, SEQ ID NO: 37.

In one embodiment the CD3 binding antigen binding moiety, particularlyFab molecule, comprises a heavy chain variable region comprising theheavy chain CDR1 of SEQ ID NO: 32, the heavy chain CDR2 of SEQ ID NO:33, the heavy chain CDR3 of SEQ ID NO: 34, and a light chain variableregion comprising the light chain CDR1 of SEQ ID NO: 35, the light chainCDR2 of SEQ ID NO: 36, and the light chain CDR3 of SEQ ID NO: 37.

In one embodiment the CD3 binding antigen binding moiety, particularlyFab molecule, comprises a heavy chain variable region sequence that isat least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:38 and a light chain variable region sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 39.

In one embodiment the CD3 binding antigen binding moiety, particularlyFab molecule, comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 38 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 39.

In one embodiment the CD3 binding antigen binding moiety, particularlyFab molecule, comprises the heavy chain variable region sequence of SEQID NO: 38 and the light chain variable region sequence of SEQ ID NO: 39.

In some embodiments, the activating T cell antigen binding moietyspecifically binds to CD3, particularly CD3 epsilon, and comprises atleast one heavy chain HVR selected from the group consisting of SEQ IDNO: 120, SEQ ID NO: 121 and SEQ ID NO: 122 and at least one light chainHVR selected from the group of SEQ ID NO: 123, SEQ ID NO: 124, SEQ IDNO: 125.

In one embodiment the CD3 binding antigen binding moiety, particularlyFab molecule, comprises a heavy chain variable region comprising theheavy chain HVR 1 (H1-HVR) of SEQ ID NO: 120, the H2-HVR of SEQ ID NO:121, and the H3-HVR of SEQ ID NO: 122; and a light chain variable regioncomprising the light chain HVR 1 (L1-HVR) of SEQ ID NO: 123, the L2-HVRof SEQ ID NO: 124 and the L3-HVR of SEQ ID NO: 125.

In one embodiment the CD3 binding antigen binding moiety, particularlyFab molecule, comprises a heavy chain variable region sequence that isat least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:126 and a light chain variable region sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 127.

In one embodiment the CD3 binding antigen binding moiety, particularlyFab molecule, comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 126 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 127.

In one embodiment the CD3 binding antigen binding moiety, particularlyFab molecule, comprises the heavy chain variable region sequence of SEQID NO: 126 and the light chain variable region sequence of SEQ ID NO:127.

(ii) Target Cell Antigen Binding Moiety

In some embodiments, an antibody comprised in the therapeutic agent,particularly a bispecific antibody, comprises at least one antigenbinding moiety, particularly a Fab molecule, which specifically binds toa target cell antigen. In certain embodiments, the antibody comprisestwo antigen binding moieties, particularly Fab molecules, whichspecifically bind to a target cell antigen. In a particular suchembodiment, each of these antigen binding moieties specifically binds tothe same antigenic determinant. In an even more particular embodiment,all of these antigen binding moieties are identical, i.e. they comprisethe same amino acid sequences including the same amino acidsubstitutions in the CH1 and CL domain as described herein (if any). Inone embodiment, the antibody comprises an immunoglobulin molecule whichspecifically binds to a target cell antigen. In one embodiment theantibody comprises not more than two antigen binding moieties,particularly Fab molecules, which specifically bind to a target cellantigen.

In particular embodiments, the antigen binding moiety(ies) whichspecifically bind to a target cell antigen is/are a conventional Fabmolecule. In such embodiments, the antigen binding moiety(ies) whichspecifically binds an activating T cell antigen is a crossover Fabmolecule as described herein, i.e. a Fab molecule wherein the variabledomains VH and VL or the constant domains CH1 and CL of the Fab heavyand light chains are exchanged/replaced by each other.

In alternative embodiments, the antigen binding moiety(ies) whichspecifically bind to a target cell antigen is/are a crossover Fabmolecule as described herein, i.e. a Fab molecule wherein the variabledomains VH and VL or the constant domains CH1 and CL of the Fab heavyand light chains are exchanged/replaced by each other. In suchembodiments, the antigen binding moiety(ies) which specifically binds anactivating T cell antigen is a conventional Fab molecule.

The target cell antigen binding moiety is able to direct the antibody toa target site, for example to a specific type of tumor cell thatexpresses the target cell antigen.

In one embodiment, the target cell antigen is CEA, particularly humanCEA.

In one embodiment, the antigen binding moiety, particularly Fabmolecule, which specifically binds to CEA comprises a heavy chainvariable region comprising the heavy chain complementarity determiningregion (CDR) 1 of SEQ ID NO: 14, the heavy chain CDR 2 of SEQ ID NO: 15,and the heavy chain CDR 3 of SEQ ID NO: 16, and a light chain variableregion comprising the light chain CDR 1 of SEQ ID NO: 17, the lightchain CDR 2 of SEQ ID NO: 18 and the light chain CDR 3 of SEQ ID NO: 19.In a further embodiment, the antigen binding moiety, particularly Fabmolecule, which specifically binds to CEA comprises a heavy chainvariable region that is at least 95%, 96%, 97%, 98%, or 99% identical tothe sequence of SEQ ID NO: 20, and a light chain variable region that isat least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNO: 21. In still a further embodiment, the antigen binding moiety,particularly Fab molecule, which specifically binds to CEA comprises theheavy chain variable region sequence of SEQ ID NO: 20, and the lightchain variable region sequence of SEQ ID NO: 21.

In one embodiment, the antigen binding moiety, particularly Fabmolecule, which specifically binds to CEA comprises a heavy chainvariable region comprising the heavy chain complementarity determiningregion (CDR) 1 of SEQ ID NO: 136, the heavy chain CDR 2 of SEQ ID NO:137, and the heavy chain CDR 3 of SEQ ID NO: 138, and a light chainvariable region comprising the light chain CDR 1 of SEQ ID NO: 139, thelight chain CDR 2 of SEQ ID NO: 140 and the light chain CDR 3 of SEQ IDNO: 141. In a further embodiment, the antigen binding moiety,particularly Fab molecule, which specifically binds to CEA comprises aheavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 142, and a light chain variableregion that is at least 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 143. In still a further embodiment, the antigenbinding moiety, particularly Fab molecule, which specifically binds toCEA comprises the heavy chain variable region sequence of SEQ ID NO:142, and the light chain variable region sequence of SEQ ID NO: 143.

In one embodiment, the target cell antigen is a B-cell antigen,particularly a malignant B-cell antigen. In one embodiment, the targetcell antigen is a cell surface antigen. In one embodiment the targetcell antigen is selected from the group consisting of CD20, CD19, CD22,ROR-1, CD37 and CD5.

In one embodiment, the target cell antigen is CD20, particularly humanCD20.

In one embodiment, the antigen binding moiety, particularly Fabmolecule, which specifically binds to CD20 comprises a heavy chainvariable region comprising the heavy chain complementarity determiningregion (CDR) 1 of SEQ ID NO: 4, the heavy chain CDR 2 of SEQ ID NO: 5,and the heavy chain CDR 3 of SEQ ID NO: 6, and a light chain variableregion comprising the light chain CDR 1 of SEQ ID NO: 7, the light chainCDR 2 of SEQ ID NO: 8 and the light chain CDR 3 of SEQ ID NO: 9. In afurther embodiment, the antigen binding moiety, particularly Fabmolecule, which specifically binds to CD20 comprises a heavy chainvariable region that is at least 95%, 96%, 97%, 98%, or 99% identical tothe sequence of SEQ ID NO: 10, and a light chain variable region that isat least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNO: 11. In still a further embodiment, the antigen binding moiety,particularly Fab molecule, which specifically binds to CD20 comprisesthe heavy chain variable region sequence of SEQ ID NO: 10, and the lightchain variable region sequence of SEQ ID NO: 11.

In one embodiment, the antigen binding moiety, particularly Fabmolecule, which specifically binds to CD20 comprises a heavy chainvariable region comprising the heavy chain HVR 1 (H1-HVR) of SEQ ID NO:128, the H2-HVR of SEQ ID NO: 129, and the H3-HVR of SEQ ID NO: 130; anda light chain variable region comprising the light chain HVR 1 (L1-HVR)of SEQ ID NO: 131, the L2-HVR of SEQ ID NO: 132 and the L3-HVR of SEQ IDNO: 133. In a further embodiment, the antigen binding moiety,particularly Fab molecule, which specifically binds to CD20 comprises aheavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 134, and a light chain variableregion that is at least 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 135. In still a further embodiment, the antigenbinding moiety, particularly Fab molecule, which specifically binds toCD20 comprises the heavy chain variable region sequence of SEQ ID NO:134, and the light chain variable region sequence of SEQ ID NO: 135.

In one embodiment, the target cell antigen is CD19, particularly humanCD19.

In one embodiment, the antigen binding moiety, particularly Fabmolecule, which specifically binds to CD19 comprises a heavy chainvariable region comprising the heavy chain complementarity determiningregion (CDR) 1 of SEQ ID NO: 48, the heavy chain CDR 2 of SEQ ID NO: 49,and the heavy chain CDR 3 of SEQ ID NO: 50, and a light chain variableregion comprising the light chain CDR 1 of SEQ ID NO: 51, the lightchain CDR 2 of SEQ ID NO: 52 and the light chain CDR 3 of SEQ ID NO: 53.In a further embodiment, the antigen binding moiety, particularly Fabmolecule, which specifically binds to CD19 comprises a heavy chainvariable region that is at least 95%, 96%, 97%, 98%, or 99% identical tothe sequence of SEQ ID NO: 54, and a light chain variable region that isat least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNO: 55. In still a further embodiment, the antigen binding moiety,particularly Fab molecule, which specifically binds to CD19 comprisesthe heavy chain variable region sequence of SEQ ID NO: 54, and the lightchain variable region sequence of SEQ ID NO: 55.

In another embodiment, the antigen binding moiety, particularly Fabmolecule, which specifically binds to CD19 comprises a heavy chainvariable region comprising the heavy chain complementarity determiningregion (CDR) 1 of SEQ ID NO: 59, the heavy chain CDR 2 of SEQ ID NO: 60,and the heavy chain CDR 3 of SEQ ID NO: 61, and a light chain variableregion comprising the light chain CDR 1 of SEQ ID NO: 62, the lightchain CDR 2 of SEQ ID NO: 63 and the light chain CDR 3 of SEQ ID NO: 64.In a further embodiment, the antigen binding moiety, particularly Fabmolecule, which specifically binds to CD19 comprises a heavy chainvariable region that is at least 95%, 96%, 97%, 98%, or 99% identical tothe sequence of SEQ ID NO: 65, and a light chain variable region that isat least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNO: 66. In still a further embodiment, the antigen binding moiety,particularly Fab molecule, which specifically binds to CD19 comprisesthe heavy chain variable region sequence of SEQ ID NO: 65, and the lightchain variable region sequence of SEQ ID NO: 66.

In another embodiment, the antigen binding moiety, particularly Fabmolecule, which specifically binds to CD19 comprises

-   -   (i) a heavy chain variable region comprising the heavy chain        complementarity determining region (CDR) 1 of SEQ ID NO: 67, the        heavy chain CDR 2 of SEQ ID NO: 68, and the heavy chain CDR 3 of        SEQ ID NO: 69, and a light chain variable region comprising the        light chain CDR 1 of SEQ ID NO: 70, the light chain CDR 2 of SEQ        ID NO: 71 and the light chain CDR 3 of SEQ ID NO: 72;    -   (ii) a heavy chain variable region comprising the heavy chain        complementarity determining region (CDR) 1 of SEQ ID NO: 75, the        heavy chain CDR 2 of SEQ ID NO: 76, and the heavy chain CDR 3 of        SEQ ID NO: 77, and a light chain variable region comprising the        light chain CDR 1 of SEQ ID NO: 78, the light chain CDR 2 of SEQ        ID NO: 79 and the light chain CDR 3 of SEQ ID NO: 80;    -   (iii) a heavy chain variable region comprising the heavy chain        complementarity determining region (CDR) 1 of SEQ ID NO: 83, the        heavy chain CDR 2 of SEQ ID NO: 84, and the heavy chain CDR 3 of        SEQ ID NO: 85, and a light chain variable region comprising the        light chain CDR 1 of SEQ ID NO: 86, the light chain CDR 2 of SEQ        ID NO: 87 and the light chain CDR 3 of SEQ ID NO: 88;    -   (iv) a heavy chain variable region comprising the heavy chain        complementarity determining region (CDR) 1 of SEQ ID NO: 91, the        heavy chain CDR 2 of SEQ ID NO: 92, and the heavy chain CDR 3 of        SEQ ID NO: 93, and a light chain variable region comprising the        light chain CDR 1 of SEQ ID NO: 94, the light chain CDR 2 of SEQ        ID NO: 95 and the light chain CDR 3 of SEQ ID NO: 96;    -   (v) a heavy chain variable region comprising the heavy chain        complementarity determining region (CDR) 1 of SEQ ID NO: 99, the        heavy chain CDR 2 of SEQ ID NO: 100, and the heavy chain CDR 3        of SEQ ID NO: 101, and a light chain variable region comprising        the light chain CDR 1 of SEQ ID NO: 102, the light chain CDR 2        of SEQ ID NO: 103 and the light chain CDR 3 of SEQ ID NO: 104;        or    -   (vi) a heavy chain variable region comprising the heavy chain        complementarity determining region (CDR) 1 of SEQ ID NO: 107,        the heavy chain CDR 2 of SEQ ID NO: 108, and the heavy chain CDR        3 of SEQ ID NO: 109, and a light chain variable region        comprising the light chain CDR 1 of SEQ ID NO: 110, the light        chain CDR 2 of SEQ ID NO: 111 and the light chain CDR 3 of SEQ        ID NO: 112.

In a further embodiment, the antigen binding moiety, particularly Fabmolecule, which specifically binds to CD19 comprises

-   -   (i) a heavy chain variable region that is at least 95%, 96%,        97%, 98%, or 99% identical to the sequence of SEQ ID NO: 73, and        a light chain variable region that is at least 95%, 96%, 97%,        98%, or 99% identical to the sequence of SEQ ID NO: 74;    -   (ii) a heavy chain variable region that is at least 95%, 96%,        97%, 98%, or 99% identical to the sequence of SEQ ID NO: 81, and        a light chain variable region that is at least 95%, 96%, 97%,        98%, or 99% identical to the sequence of SEQ ID NO: 82;    -   (iii) a heavy chain variable region that is at least 95%, 96%,        97%, 98%, or 99% identical to the sequence of SEQ ID NO: 89, and        a light chain variable region that is at least 95%, 96%, 97%,        98%, or 99% identical to the sequence of SEQ ID NO: 90;    -   (iv) a heavy chain variable region that is at least 95%, 96%,        97%, 98%, or 99% identical to the sequence of SEQ ID NO: 97, and        a light chain variable region that is at least 95%, 96%, 97%,        98%, or 99% identical to the sequence of SEQ ID NO: 98;    -   (v) a heavy chain variable region that is at least 95%, 96%,        97%, 98%, or 99% identical to the sequence of SEQ ID NO: 105,        and a light chain variable region that is at least 95%, 96%,        97%, 98%, or 99% identical to the sequence of SEQ ID NO: 106; or    -   (vi) a heavy chain variable region that is at least 95%, 96%,        97%, 98%, or 99% identical to the sequence of SEQ ID NO: 113,        and a light chain variable region that is at least 95%, 96%,        97%, 98%, or 99% identical to the sequence of SEQ ID NO: 114.

In still a further embodiment, the antigen binding moiety, particularlyFab molecule, which specifically binds to CD19 comprises

-   -   (i) the heavy chain variable region sequence of SEQ ID NO: 73,        and the light chain variable region sequence of SEQ ID NO: 74;    -   (ii) the heavy chain variable region sequence of SEQ ID NO: 81,        and the light chain variable region sequence of SEQ ID NO: 82;    -   (iii) the heavy chain variable region sequence of SEQ ID NO: 89,        and the light chain variable region sequence of SEQ ID NO: 90;    -   (iv) the heavy chain variable region sequence of SEQ ID NO: 97,        and the light chain variable region sequence of SEQ ID NO: 98;    -   (v) the heavy chain variable region sequence of SEQ ID NO: 105,        and the light chain variable region sequence of SEQ ID NO: 106;        or    -   (vi) the heavy chain variable region sequence of SEQ ID NO: 113,        and the light chain variable region sequence of SEQ ID NO: 114.

Charge Modifications

An antibody, particularly a multispecific antibody, comprised in thetherapeutic agent may comprise amino acid substitutions in Fab moleculescomprised therein which are particularly efficient in reducingmispairing of light chains with non-matching heavy chains(Bence-Jones-type side products), which can occur in the production ofFab-based bi-/multispecific antigen binding molecules with a VH/VLexchange in one (or more, in case of molecules comprising more than twoantigen-binding Fab molecules) of their binding arms (see also PCTpublication no. WO 2015/150447, particularly the examples therein,incorporated herein by reference in its entirety).

Accordingly, in particular embodiments, an antibody comprised in thetherapeutic agent comprises

-   -   (a) a first Fab molecule which specifically binds to a first        antigen    -   (b) a second Fab molecule which specifically binds to a second        antigen, and wherein the variable domains VL and VH of the Fab        light chain and the Fab heavy chain are replaced by each other,    -   wherein the first antigen is an activating T cell antigen and        the second antigen is a target cell antigen, or the first        antigen is a target cell antigen and the second antigen is an        activating T cell antigen; and    -   wherein    -   i) in the constant domain CL of the first Fab molecule under a)        the amino acid at position 124 is substituted by a positively        charged amino acid (numbering according to Kabat), and wherein        in the constant domain CH1 of the first Fab molecule under a)        the amino acid at position 147 or the amino acid at position 213        is substituted by a negatively charged amino acid (numbering        according to Kabat EU index); or    -   ii) in the constant domain CL of the second Fab molecule        under b) the amino acid at position 124 is substituted by a        positively charged amino acid (numbering according to Kabat),        and wherein in the constant domain CH1 of the second Fab        molecule under b) the amino acid at position 147 or the amino        acid at position 213 is substituted by a negatively charged        amino acid (numbering according to Kabat EU index).

The antibody does not comprise both modifications mentioned under i) andii). The constant domains CL and CH1 of the second Fab molecule are notreplaced by each other (i.e. remain unexchanged).

In one embodiment of the antibody, in the constant domain CL of thefirst Fab molecule under a) the amino acid at position 124 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat) (in one preferred embodimentindependently by lysine (K) or arginine (R)), and in the constant domainCH1 of the first Fab molecule under a) the amino acid at position 147 orthe amino acid at position 213 is substituted independently by glutamicacid (E), or aspartic acid (D) (numbering according to Kabat EU index).

In a further embodiment, in the constant domain CL of the first Fabmolecule under a) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the first Fabmolecule under a) the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In a particular embodiment, in the constant domain CL of the first Fabmolecule under a) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)) and the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)), and in the constant domain CH1 of the first Fabmolecule under a) the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

In a more particular embodiment, in the constant domain CL of the firstFab molecule under a) the amino acid at position 124 is substituted bylysine (K) (numbering according to Kabat) and the amino acid at position123 is substituted by lysine (K) or arginine (R) (numbering according toKabat), and in the constant domain CH1 of the first Fab molecule undera) the amino acid at position 147 is substituted by glutamic acid (E)(numbering according to Kabat EU index) and the amino acid at position213 is substituted by glutamic acid (E) (numbering according to Kabat EUindex).

In an even more particular embodiment, in the constant domain CL of thefirst Fab molecule under a) the amino acid at position 124 issubstituted by lysine (K) (numbering according to Kabat) and the aminoacid at position 123 is substituted by arginine (R) (numbering accordingto Kabat), and in the constant domain CH1 of the first Fab moleculeunder a) the amino acid at position 147 is substituted by glutamic acid(E) (numbering according to Kabat EU index) and the amino acid atposition 213 is substituted by glutamic acid (E) (numbering according toKabat EU index).

In particular embodiments, the constant domain CL of the first Fabmolecule under a) is of kappa isotype.

Alternatively, the amino acid substitutions according to the aboveembodiments may be made in the constant domain CL and the constantdomain CH1 of the second Fab molecule under b) instead of in theconstant domain CL and the constant domain CH1 of the first Fab moleculeunder a). In particular such embodiments, the constant domain CL of thesecond Fab molecule under b) is of kappa isotype.

The antibody may further comprise a third Fab molecule whichspecifically binds to the first antigen. In particular embodiments, saidthird Fab molecule is identical to the first Fab molecule under a). Inthese embodiments, the amino acid substitutions according to the aboveembodiments will be made in the constant domain CL and the constantdomain CH1 of each of the first Fab molecule and the third Fab molecule.Alternatively, the amino acid substitutions according to the aboveembodiments may be made in the constant domain CL and the constantdomain CH1 of the second Fab molecule under b), but not in the constantdomain CL and the constant domain CH1 of the first Fab molecule and thethird Fab molecule.

In particular embodiments, the antibody further comprises an Fc domaincomposed of a first and a second subunit capable of stable association.

Treatment Regimen

According to the invention, the Type II anti-CD20 antibody and thetherapeutic agent may be administered in various ways (e.g. with regardto the route of administration, dose and/or timing), as long as the TypeII anti-CD20 antibody is administered prior to the therapeutic agent andthat the administration of the Type II anti-CD20 antibody haseffectively induced a reduction of the number of B cells in the treatedsubject by the time the therapeutic agent is administered.

Without wishing to be bound by theory, the reduction of the number of Bcells in the subject prior to administration of the therapeutic agentwill reduce or prevent the formation of anti-drug antibodies (ADAs) tothe therapeutic agent and thus reduce or prevent a loss of efficacy ofthe therapeutic agent and/or adverse events in the subject associatedwith ADAs, and/or will reduce or prevent cytokine release associatedwith administration of the therapeutic agent and thus reduce or preventadverse events (such as IRRs) in the subject associated with theadministration of the therapeutic agent.

In one embodiment, the treatment regimen effectively reduces theformation of anti-drug antibodies (ADAs) in the subject in response tothe administration of the therapeutic agent as compared to acorresponding treatment regimen without the administration of the TypeII anti-CD20 antibody. In one embodiment, the formation of ADAs isreduced at least 2-fold, at least 3-fold, at least 4-fold, at least5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or atleast 100-fold as compared to a corresponding treatment regimen withoutthe administration of the Type II anti-CD20 antibody. In one embodiment,the formation of ADAs is essentially prevented. In one embodiment, thereduction or prevention of the formation of ADAs is 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, or 1 month,2 months, 3 months, 4 months, 5 months, 6 months, 12 months, or more,after administration of the therapeutic agent. In one embodiment, thereduction or prevention of ADA is about 2 months after administration ofthe therapeutic agent.

In one embodiment, the ADA titer in the subject after administration ofthe therapeutic agent does not exceed the ADA titer in the subject priorto administration of the therapeutic agent. In one embodiment, the ADAtiter in the subject after administration of the therapeutic agent doesnot exceed the ADA titer in the subject prior to administration of thetherapeutic agent by more than 1.1-fold, more than 1.2-fold, more than1.5-fold, more than 2-fold, more than 3-fold, more than 4-fold, morethan 5-fold, or more than 10-fold. In one embodiment, the ADA titer inthe subject after administration of the therapeutic agent is increasedless than 1.1-fold, less than 1.2-fold, less than 1.5-fold, less than2-fold, less than 3-fold, less than 4-fold, less than 5-fold, or lessthan 10-fold, as compared to the ADA titer in the subject prior toadministration of the therapeutic agent. In one embodiment, the ADAtiter in the subject after administration of the therapeutic agent isthe ADA titer at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or 21 days, or 1 month, 2 months, 3 months, 4 months, 5months, 6 months, 12 months, or more, after administration of thetherapeutic agent. In one embodiment, the ADA titer in the subject afteradministration of the therapeutic agent is the ADA titer at about 2months after administration of the therapeutic agent.

In one embodiment, essentially no ADAs are detectable in the subjectafter administration of the therapeutic agent, particularly 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, or1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, ormore, after administration of the therapeutic agent.

ADAs can be detected by methods known in the art (see e.g. Mire-Sluis etal., J Immunol Methods (2004) 289, 1-16; Nencini et al., Drug Dev Res(2014), 75 Suppl 1, S4-6; Schouwenburg et al., Nat Rev Rheumatol (2015)9, 164-172). An exemplary method to detect ADAs is a sandwich ELISA, inwhich the therapeutic agent (e.g. an antibody) is coated to the assayplate, is exposed to serum of the treated subject, and the presence ofADAs is detected by labelled therapeutic agent. Another exemplary methodto detect ADAs is an antigen binding test wherein immunoglobulins fromthe treated subject's serum are aggregated on a protein (e.g. Protein ASepharose) and the presence of ADAs is detected by labelled therapeuticagent.

ADAs can be detected e.g. in a blood sample taken from the subject. Inone embodiment, the ADA titer in the subject (as measured e.g. in ablood sample taken from the subject) does not exceed a titer (i.e.highest possible dilution of the sample giving an assay signal above theassay cut point) of about 10, of about 20, of about 30, of about 40, ofabout 50, of about 100, of about 200, or about 500, or of about 1000after the administration of the therapeutic agent, particularly 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days,or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months,or more, after administration of the therapeutic agent.

In some embodiments, the treatment regimen increases efficacy of thetherapeutic agent, as compared to a corresponding treatment regimenwithout the administration of the Type II anti-CD20 antibody. In someembodiments, the treatment regimen increases overall survival of thesubject, as compared to a corresponding treatment regimen without theadministration of the Type II anti-CD20 antibody. In some embodiments,the treatment regimen increases progression-free survival of thesubject, as compared to a corresponding treatment regimen without theadministration of the Type II anti-CD20 antibody.

In one embodiment, the treatment regimen effectively reduces cytokinerelease in the subject associated with the administration of thetherapeutic agent as compared to a corresponding treatment regimenwithout the administration of the Type II anti-CD20 antibody. In oneembodiment, cytokine release is reduced at least 2-fold, at least3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least20-fold, at least 50-fold, or at least 100-fold as compared to acorresponding treatment regimen without the administration of the TypeII anti-CD20 antibody. In one embodiment, cytokine release isessentially prevented. In one embodiment, the reduction or prevention ofcytokine release is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23 or 24 hours after administration of thetherapeutic agent. In one embodiment, the reduction or prevention ofcytokine release is within the first 24 hours after administration ofthe therapeutic agent.

In one embodiment, the cytokine concentration in the subject (asmeasured e.g. in a blood sample taken from the subject) afteradministration of the therapeutic agent does not exceed the cytokineconcentration in the subject prior to administration of the therapeuticagent. In one embodiment, the cytokine concentration in the subjectafter administration of the therapeutic agent does not exceed thecytokine concentration in the subject prior to administration of thetherapeutic agent by more than 1.1-fold, more than 1.2-fold, more than1.5-fold, more than 2-fold, more than 3-fold, more than 4-fold, morethan 5-fold, more than 10-fold, more than 20-fold, more than 50-fold ormore than 100-fold. In one embodiment, the cytokine concentration in thesubject after administration of the therapeutic agent is increased lessthan 1.1-fold, less than 1.2-fold, less than 1.5-fold, less than 2-fold,less than 3-fold, less than 4-fold, less than 5-fold, less than 10-fold,less than 20-fold, less than 50-fold or less than 100-fold, as comparedto the cytokine concentration in the subject prior to administration ofthe therapeutic agent. In one embodiment, the cytokine concentration inthe subject after administration of the therapeutic agent is thecytokine concentration at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours after administration ofthe therapeutic agent. In one embodiment, the cytokine concentration inthe subject after administration of the therapeutic agent is thecytokine concentration within the first 24 hours after administration ofthe therapeutic agent.

In one embodiment, essentially no increase in the concentration ofcytokines is detectable in the subject after administration of thetherapeutic agent, particularly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours afteradministration of the therapeutic agent.

Cytokines can be detected by methods known in the art, such as e.g.ELISA, FACS or Luminex® assay.

Cytokines can be detected e.g. in a blood sample taken from the subject.In one embodiment, the cytokine concentration is the blood of thesubject.

In some embodiments, the cytokine is one or more cytokine(s) selectedfrom the group consisting of tumor necrosis factor alpha (TNF-α),interferon gamma (IFN-γ), interleukin-6 (IL-6), interleukin-10 (IL-10),interleukin-2 (IL-2) and interleukin-8 (IL-8), particularly the groupconsisting of TNF-α, IFN-γ and IL-6. In some embodiments, the cytokineis TNF-α. In some embodiments, the cytokine is IFN-γ. In someembodiments, the cytokine is IL-6. In some embodiments, the cytokine isIL-10. In some embodiments, the cytokine is IL-2. In some embodiments,the cytokine is IL-8.

In some embodiments, the treatment regimen increases the safety of thetherapeutic agent, as compared to a corresponding treatment regimenwithout the administration of the Type II anti-CD20 antibody. In someembodiments, the treatment regimen reduces adverse events in thesubject, as compared to a corresponding treatment regimen without theadministration of the Type II anti-CD20 antibody. In some embodiments,the treatment regimen increases the serum half-life of the therapeuticagent, as compared to a corresponding treatment regimen without theadministration of the Type II anti-CD20 antibody. In some embodiments,the treatment regimen reduces toxicity of the therapeutic agent, ascompared to a corresponding treatment regimen without the administrationof the Type II anti-CD20 antibody.

According to the invention, the period of time between theadministration of the Type II anti-CD20 antibody and the administrationof the therapeutic agent is sufficient for reduction of the number ofB-cells in the subject in response to the administration of the Type IIanti-CD20 antibody.

In one embodiment, the period of time is 3 days to 21 days, 5 days to 20days, 7 days to 21 days, 7 days to 14 days, 5 days to 15 days, 7 days to15 days, 8 days to 15 days, 10 days to 20 days, 10 days to 15 days, 11days to 14 days, or 12 days to 13 days. In one embodiment, the period oftime is 7 days to 14 days. In a particular embodiment, the period oftime is 10 days to 15 days. In one embodiment, the period of time is 8days to 15 days. In one embodiment, the period of time is 5 days to 10days. In a particular embodiment, the period of time is 7 days.

In one embodiment, the period of time is about 3 days, about 4 days,about 5 days, about 6 days, about 7 days, about 8 days, about 9 days,about 10 days, about 11 days, about 12 days, about 13 days, about 14days, about 15 days, about 16 days, about 17 days, about 18 days, about19 days, about 20 days, about 21 days, about 22 days, about 23 days,about 24 days, about 25 days, about 26 days, about 27 days, about 28days, about 29 days, or about 30 days.

In one embodiment, the period of time is at least 3 days, at least 4days, at least 5 days, at least 6 days, at least 7 days, at least 8days, at least 9 days, at least 10 days, at least 11 days, at least 12days, at least 13 days, at least 14 days, or at least 15 days. In aparticular embodiment, the period of time is at least 5 days. In afurther particular embodiment, the period of time is at least 7 days.

In one embodiment, the period of time is between the last administrationof the Type II anti-CD20 antibody and the (first, if several)administration of the therapeutic agent. In one embodiment, noadministration of the therapeutic agent is made during the period oftime.

In a particular embodiment, the reduction of the number of B cells is inthe blood of the subject. In one embodiment, the B cells are peripheralblood B cells. In one embodiment, the B cells are normal B cells. In oneembodiment, the B cells are malignant and normal B cells. In oneembodiment, the B cells are malignant B cells.

In some embodiments, the reduction of B cells is in a tissue of thesubject. In one embodiment, the tissue is a tumor. In one embodiment,the tissue is a lymph node. In one embodiment, the tissue is spleen. Inone embodiment, the tissue is the marginal zone of spleen. In oneembodiment, the B cells are lymph node B cells. In one embodiment, the Bcells are splenic B cells. In one embodiment, the B cells are splenicmarginal zone B cells. In one embodiment, the B cells are CD20-positiveB cells, i.e. B cells expressing CD20 on their surface.

In one embodiment, the reduction of the number of B cells is a reductionof at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, or at leastabout 95%. In one embodiment, the reduction of the number of B cells isa complete elimination of B cells. In a particular embodiment, thereduction of the number of B cells is a reduction of at least 90%,particularly at least 95%, of the number of B cells in the (peripheral)blood of the subject. In one embodiment, the reduction of the number ofB cells is a reduction as compared to the number of B cells in thesubject prior to the (first, if several) administration of the Type IIanti-CD20 antibody to the subject.

The number of B cells in the subject may be determined by any methodknown in the art suitable for quantifying B cells in patient blood ortissue, such as flow cytometric, immunohistochemical orimmunofluorescent methods, using antibodies against B cell markers suchas CD20, CD19, and/or PAX5.

The number of B cells may also be determined indirectly, byquantification of protein or mRNA levels of B-cell markers in patientblood or tissues. Suitable methods known in the art for thedetermination of specific protein levels include immunoassay methodssuch as enzyme-linked immunosorbent assay (ELISA), or Western Blot,methods for determination of mRNA levels include for examplequantitative RT-PCR or microarray technologies.

All the above mentioned methods and technologies are well known in theart and can be deduced from standard textbooks such as Lottspeich(Bioanalytik, Spektrum Akademisher Verlag, 1998) or Sambrook and Russell(Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor,NY, U.S.A., 2001).

In certain embodiments, the reduction of the number of B cells isdetermined by quantification of B cells in the blood of the subject(e.g. in a blood sample taken from the subject). In one such embodiment,B cells are quantified by flow cytometric analysis. Flow cytometricmethods (FACS) are well known in the art for the quantification of cellsin blood or tissue samples. In particular, they allow determining thenumber of cells expressing a specific antigen (e.g. CD20 and/or CD19)among a defined total number of cells in a blood or tissue sample (e.g.a blood sample, or (part of) a tissue biopsy). In one embodiment, Bcells are quantified by flow cytometric analysis using an anti-CD19antibody and/or an anti-CD20 antibody.

In other embodiments, the reduction of the number of B cells isdetermined by quantification of B cells in a tissue, e.g. a tumor, ofsaid individual (e.g. in a tissue biopsy taken from the subject). In onesuch embodiment, B cells are quantified by immunohistochemical orimmunofluorescent analysis. In one embodiment, B cells are quantified byimmunohistochemical analysis using an anti-CD19 antibody, an anti-CD20antibody and/or an anti-PAX5 antibody.

Methods of the present invention can be applied in the treatment of avariety of diseases, depending on the therapeutic agent(s) used.

In certain embodiments the disease to be treated is a proliferativedisorder, particularly cancer. Non-limiting examples of cancers includebladder cancer, brain cancer, head and neck cancer, pancreatic cancer,lung cancer, breast cancer, ovarian cancer, uterine cancer, cervicalcancer, endometrial cancer, esophageal cancer, colon cancer, colorectalcancer, rectal cancer, gastric cancer, prostate cancer, blood cancer,skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.Other cell proliferation disorders that can be treated using a method ofthe present invention include, but are not limited to neoplasms locatedin the: abdomen, bone, breast, digestive system, liver, pancreas,peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous system(central and peripheral), lymphatic system, pelvic, skin, soft tissue,spleen, thoracic region, and urogenital system. Also included arepre-cancerous conditions or lesions and cancer metastases. In certainembodiments the cancer is chosen from the group consisting of renal cellcancer, skin cancer, lung cancer, colorectal cancer, breast cancer,brain cancer, head and neck cancer. In some embodiments, the cancer is asolid tumor. In some embodiments, the cancer expresses the target of thetherapeutic agent, e.g. an antibody. In one embodiment, the cancerexpresses CEA. In another embodiment, the cancer expresses FAP.

In some embodiments, the disease to be treated is an inflammatorydisorder. In some embodiments, the disease to be treated is anautoimmune disease (i.e. a non-malignant disease or disorder arisingfrom and directed against an individual's own tissues). Examples ofautoimmune diseases or disorders include, but are not limited to,inflammatory responses such as inflammatory skin diseases includingpsoriasis and dermatitis (e.g. atopic dermatitis); responses associatedwith inflammatory bowel disease (such as Crohn's disease and ulcerativecolitis); dermatitis; allergic conditions such as eczema and asthma;rheumatoid arthritis; systemic lupus erythematosus (SLE) (including butnot limited to lupus nephritis, cutaneous lupus); diabetes mellitus(e.g. type 1 diabetes mellitus or insulin dependent diabetes mellitus);multiple sclerosis and juvenile onset diabetes. In one embodiment, thedisease is transplant rejection or graft-versus-host disease.

In some embodiments, the disease to be treated is an infectious disease,such as viral infection or a bacterial infection. In other embodiments,the disease to be treated is neurological disorder. In still furtherembodiments, the disease to be treated is a metabolic disorder.

In relation aspects of the invention concerned with the reduction ofcytokine release associated with the administration of a therapeuticagent in a subject, the methods are particularly useful, in thetreatment of B-cell proliferative disorders, particularly CD20-positiveB-cell disorders, where (CD20-positive) B-cells are present in largequantities (i.e. an increased number of B-cells is present in thesubject suffering from the disorder, as compared to a healthy subject).

Thus, in one embodiment, the disease is a B cell proliferative disorder,particularly a CD20-positive B-cell disorder.

In one embodiment, the disease is selected from the group consisting ofNon-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL), chroniclymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL),follicular lymphoma (FL), mantle-cell lymphoma (MCL), marginal zonelymphoma (MZL), Multiple myeloma (MM) or Hodgkin lymphoma (HL). In oneembodiment, the disease is selected from the group consisting ofNon-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL), chroniclymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL),follicular lymphoma (FL), mantle-cell lymphoma (MCL) and marginal zonelymphoma (MZL).

In a particular embodiment, the disease is NHL, particularlyrelapsed/refractory (r/r) NHL. In one embodiment, the disease is DLBCL.In one embodiment, the disease is FL. In one embodiment, the disease isMCL. In one embodiment, the disease is MZL A skilled artisan readilyrecognizes that in many cases the therapeutic agent may not provide acure but may only provide partial benefit. In some embodiments, aphysiological change having some benefit is also consideredtherapeutically beneficial. Thus, in some embodiments, an amount oftherapeutic agent that provides a physiological change is considered an“effective amount” or a “therapeutically effective amount”.

The subject, patient, or individual in need of treatment is typically amammal, more specifically a human. In certain embodiments, the subjectis a human.

In some embodiments, in particular in relation aspects of the inventionconcerned with the reduction of the formation of anti-drug antibodies(ADAs) against a therapeutic agent in a subject, the subject suffersfrom a locally advanced and/or metastatic solid tumor and has progressedon or is intolerant to the standard of care therapy. In one embodiment,the tumor is a CEA-expressing tumor. In another embodiment, the tumor isa FAP-expressing tumor.

Expression of CEA and/or FAP in a tumor may be determined for example byimmunohistochemical analysis of a tumor biopsy taken from the subject.

In other embodiments, in particular in relation aspects of the inventionconcerned with the reduction of cytokine release associated with theadministration of a therapeutic agent in a subject, the subject suffersfrom a B-cell proliferative disorder, particularly from Non-Hodgkinlymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocyticleukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicularlymphoma (FL), mantle-cell lymphoma (MCL), marginal zone lymphoma (MZL),Multiple myeloma (MM) or Hodgkin lymphoma (HL). In one embodiment, thesubject suffers from relapsed/refractory (r/r) NHL.

Administration of the Type II Anti-CD20 Antibody

According to the invention, the period of time between theadministration of the Type II anti-CD20 antibody and the administrationof the therapeutic agent and the dose of the Type II anti-CD20 antibodyare chosen such as to effectively reduce the number of B cells in thesubject prior to administration of the therapeutic agent.

The Type II anti-CD20 antibody can be administered by any suitablemeans, including parenteral, intrapulmonary, and intranasal, and, ifdesired for local treatment, intralesional administration. Parenteralinfusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. Dosing can be by anysuitable route, e.g. by injections, such as intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic. Various dosing schedules including but not limited to single ormultiple administrations over various time-points, bolus administration,and pulse infusion are contemplated herein. In one embodiment, the TypeII anti-CD20 antibody is administered parenterally, particularlyintravenously, e.g. by intravenous infusion.

The Type II anti-CD20 antibody would be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

In one embodiment, the administration of the Type II anti-CD20 antibodyis a single administration. In another embodiment, the administration ofthe Type II anti-CD20 antibody is two or more separate administrations.In one embodiment, the two or more separate administrations are on twoor more consecutive days. In one embodiment, no further administrationof the Type II anti-CD20 antibody is made to the subject before or afterthe administration of the therapeutic agent. In one embodiment, theadministration of the Type II anti-CD20 antibody is a singleadministration, or two administrations on two consecutive days, and nofurther administration of the Type II anti-CD20 antibody is made. In oneembodiment, the period of time is between the last administration of theType II anti-CD20 antibody and the (first, if several) administration ofthe therapeutic agent.

In one embodiment, the administration of the Type II anti-CD20 antibodyis a dose of Type II anti-CD20 antibody effective for the reduction of Bcells in the subject. In one embodiment, the dose of Type II anti-CD20antibody is effective in reducing the number of B cells in the subjectwithin the period of time between the administration of the Type IIanti-CD20 antibody and the administration of the therapeutic agent. Inone embodiment, the period of time between the administration of theType II anti-CD20 antibody and the administration of the therapeuticagent and the administered dose of Type II anti-CD20 antibody issufficient for reduction of the number of B-cells in the subject inresponse to the administration of the Type II anti-CD20 antibody.

In one embodiment, the administration of the Type II anti-CD20 antibodyis a dose of about 2 g Type II anti-CD20 antibody. The dose of about 2 gType II anti-CD20 antibody may be administered to the subject as asingle administration of about 2 g, or as several administrations, e.g.two administrations of about 1 g each or three administrations of e. g.100 mg, 900 mg and 1000 mg. In one embodiment, one administration ofabout 2 g Type II anti-CD20 antibody is made to the subject. In anotherembodiment, two administrations of about 1 g Type II anti-CD20 antibodyeach are made to the subject on two consecutive days. In still anotherembodiment, three administrations ((i) to (iii)) of (i) about 100 mgType II anti-CD20 antibody, (ii) about 900 mg Type II anti-CD20antibody, and (iii) about 1000 mg Type II anti-CD20 antibody are made tothe subject on three consecutive days. In one embodiment, twoadministration of about 1 g Type II anti-CD20 antibody are made to thesubject on two consecutive days, 10 days to 15 days before theadministration of the therapeutic agent. In one embodiment, oneadministration of about 2 g Type II anti-CD20 antibody is made to thesubject 10 days to 15 days before the administration of the therapeuticagent. In one embodiment, no further administration of the Type IIanti-CD20 antibody is made to the subject. In one embodiment, noadministration of the therapeutic agent is made to the subject prior tothe administration of the Type II anti-CD20 antibody (at least notwithin the same course of treatment).

In one embodiment, the administration of the Type II anti-CD20 antibodyis a dose of about 1000 mg Type II anti-CD20 antibody. The dose of about1000 mg Type II anti-CD20 antibody may be administered to the subject asa single administration of about 1000 mg, or as several administrations,e.g. two administrations of about 500 mg each. In a particularembodiment, one administration of about 1000 mg Type II anti-CD20antibody is made to the subject. In another embodiment, twoadministrations of about 500 mg Type II anti-CD20 antibody each are madeto the subject on two consecutive days. In one embodiment, oneadministration of about 1000 mg Type II anti-CD20 antibody is made tothe subject, 7 days before the administration of the therapeutic agent.In one embodiment, no further administration of the Type II anti-CD20antibody is made to the subject. In one embodiment, no administration ofthe therapeutic agent is made to the subject prior to the administrationof the Type II anti-CD20 antibody (at least not within the same courseof treatment).

In one embodiment, the treatment regimen further comprisesadministration of premedication prior to the administration of the TypeII anti-CD20 antibody. In embodiment the premedication comprises acorticosteroid (such as e.g. prednisolone, dexamethasone, ormethylprednisolone), paracetamol/acetaminophen, and/or an anti-histamine(such as e.g. diphenhydramine). In one embodiment, the premedication isadministered at least 60 minutes prior to the administration of the TypeII anti-CD20 antibody.

In one embodiment, the treatment regimen does not compriseadministration of an immunosuppressive agent other than the Type IIanti-CD20 antibody (and optionally the above-described premedication)prior to the administration of the therapeutic agent. In one embodiment,the treatment regimen does not comprise administration of an agentselected from the group of methotrexate, azathioprine, 6-mercaptopurine,leflunomide, cyclosporine, tacrolimus/FK506, mycophenolate mofetil andmycophenolate sodium prior to the administration of the therapeuticagent. In one embodiment, the treatment regimen does not compriseadministration of a further antibody in addition to the Type IIanti-CD20 antibody prior to the administration of the therapeutic agent.

Administration of the Therapeutic Agent

The therapeutic agent can be administered by any suitable means,including parenteral, intrapulmonary, and intranasal, and, if desiredfor local treatment, intralesional administration. The methods of thepresent invention are particularly useful, however, in relation totherapeutic agents administered by parenteral, particularly intravenous,infusion. Parenteral infusions include intramuscular, intravenous,intraarterial, intraperitoneal, or subcutaneous administration. Dosingcan be by any suitable route, e.g. by injections, such as intravenous orsubcutaneous injections, depending in part on whether the administrationis brief or chronic. Various dosing schedules including but not limitedto single or multiple administrations over various time-points, bolusadministration, and pulse infusion are contemplated herein. In oneembodiment, the therapeutic agent is administered parenterally,particularly intravenously. In a particular embodiment, the therapeuticagent is administered by intravenous infusion.

The therapeutic agent would be formulated, dosed, and administered in afashion consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Thetherapeutic agent need not be, but is optionally formulated with one ormore agents currently used to prevent or treat the disorder in question.The effective amount of such other agents depends on the amount oftherapeutic agent present in the formulation, the type of disorder ortreatment, and other factors discussed above. These are generally usedin the same dosages and with administration routes as described herein,or about from 1 to 99% of the dosages described herein, or in any dosageand by any route that is empirically/clinically determined to beappropriate.

For the prevention or treatment of disease, the appropriate dosage ofthe therapeutic agent (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of therapeutic agent, the severity andcourse of the disease, whether the therapeutic agent is administered forpreventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the therapeutic agent, and thediscretion of the attending physician. The therapeutic agent is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of therapeutic agent can be an initialcandidate dosage for administration to the subject, whether, forexample, by one or more separate administrations, or by continuousinfusion. One typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment would generally be sustained until a desired suppressionof disease symptoms occurs. One exemplary dosage of the therapeuticagent would be in the range from about 0.05 mg/kg to about 10 mg/kg.Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10mg/kg (or any combination thereof) may be administered to the subject.Such doses may be administered intermittently, e.g. every week, everytwo weeks, or every three weeks (e.g. such that the subject receivesfrom about two to about twenty, or e.g. about six doses of thetherapeutic agent). An initial higher loading dose, followed by one ormore lower doses, or an initial lower dose, followed by one or morehigher doses may be administered. An exemplary dosing regimen comprisesadministering an initial dose of about 10 mg, followed by a bi-weeklydose of about 20 mg of the therapeutic agent. However, other dosageregimens may be useful. The progress of this therapy is easily monitoredby conventional techniques and assays.

In one embodiment, the administration of the therapeutic agent is asingle administration. In certain embodiments, the administration of thetherapeutic agent is two or more administrations. In one suchembodiment, the therapeutic agent is administered every week, every twoweeks, or every three weeks, particularly every two weeks. In oneembodiment, the therapeutic agent is administered in a therapeuticallyeffective amount. In one embodiment the therapeutic agent isadministered at a dose of 10 mg-20 mg. In one embodiment theadministration of the therapeutic agent comprises an initialadministration of a dose of about 10 mg therapeutic agent, and one ormore subsequent administrations of a dose of about 20 mg therapeuticagent. In one embodiment the therapeutic agent is administered at a doseof about 50 μg/kg, about 100 μg/kg, about 200 μg/kg, about 300 μg/kg,about 400 μg/kg, about 500 μg/kg, about 600 μg/kg, about 700 μg/kg,about 800 μg/kg, about 900 μg/kg or about 1000 μg/kg. In one embodiment,the therapeutic agent is administered at a dose which is higher than thedose of the therapeutic agent in a corresponding treatment regimenwithout the administration of the Type II anti-CD20 antibody. In oneembodiment the administration of the therapeutic agent comprises aninitial administration of a first dose of the therapeutic agent, and oneor more subsequent administrations of a second dose the therapeuticagent, wherein the second dose is higher than the first dose. In oneembodiment, the administration of the therapeutic agent comprises aninitial administration of a first dose of the therapeutic agent, and oneor more subsequent administrations of a second dose the therapeuticagent, wherein the first dose is not lower than the second dose.

In one embodiment, the administration of the therapeutic agent in thetreatment regimen according to the invention is the first administrationof that therapeutic agent to the subject (at least within the samecourse of treatment). In one embodiment, no administration of thetherapeutic agent is made to the subject prior to the administration ofthe Type II anti-CD20 antibody.

In the present invention, the therapeutic agent can be used either aloneor in combination with other agents in a therapy. For instance, thetherapeutic agent may be co-administered with at least one additionaltherapeutic agent. In certain embodiments, an additional therapeuticagent is an immunotherapeutic agent. In some embodiment, the additionaltherapeutic agent comprises an agent as described herein in relation tothe therapeutic agent. The invention is particularly useful in relationto combinations of several therapeutic agents which may induce theformation of ADAs or cytokine release in a subject when used in atreatment regimen without the administration of the Type II anti-CD20antibody. Such combinations may include various therapeutic agents asdescribed herein, and may particularly include one or moreimmunotherapeutic agents, e.g. for the treatment of cancer (cancerimmunotherapy). Such combination therapies noted above encompasscombined administration (where two or more therapeutic agents areincluded in the same or separate formulations), and separateadministration, in which case, administration of the therapeutic agentcan occur prior to, simultaneously, and/or following, administration ofan additional therapeutic agent or agents. In one embodiment,administration of the therapeutic agent and administration of anadditional therapeutic agent occur within about one month, or withinabout one, two or three weeks, or within about one, two, three, four,five, or six days, of each other.

Articles of Manufacture

In another aspect of the invention, an article of manufacture, e.g. akit, is provided, containing materials useful for the treatment,prevention and/or diagnosis of a disease, or for the reduction of theformation of anti-drug antibodies (ADAs) and/or the reduction ofcytokine release as described herein. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition, or holds a composition which is effective for reducing theformation of ADAs and/or cytokine release, and may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Atleast one active agent in the composition is a Type II anti-CD20antibody or a therapeutic agent as described herein. The label orpackage insert indicates that the composition is used for treating thecondition of choice and/or to reduce the formation of ADAs and/orcytokine release. Moreover, the article of manufacture may comprise (a)a first container with a composition contained therein, wherein thecomposition comprises a Type II anti-CD20 antibody as described herein;and (b) a second container with a composition contained therein, whereinthe composition comprises a therapeutic agent as described herein. Thearticle of manufacture in this embodiment of the invention may furthercomprise a package insert indicating that the compositions can be usedto treat a particular condition and/or to reduce the formation of ADAsand/or cytokine release. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

EMBODIMENTS

In the following, some of the embodiments of the invention are listed.

1. A method of treating a disease in a subject, the method comprising atreatment regimen comprising

-   -   (i) administration to the subject of a Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (ii) administration to the subject of a therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent is sufficient for reduction of the number of        B-cells in the subject in response to the administration of the        Type II anti-CD20 antibody.

2. The method of embodiment 1, wherein the treatment regimen effectivelyreduces the formation of anti-drug antibodies (ADAs) in the subject inresponse to the administration of the therapeutic agent as compared to acorresponding treatment regimen without the administration of the TypeII anti-CD20 antibody.

3. A method for reducing the formation of anti-drug antibodies (ADAs)against a therapeutic agent in a subject, comprising administration of aType II anti-CD20 antibody to the subject prior to administration of thetherapeutic agent.

4. The method of embodiment 3, wherein the period of time between theadministration of the Type II anti-CD20 antibody and administration ofthe therapeutic agent is sufficient for reduction of the number ofB-cells in the subject in response to the administration of the Type IIanti-CD20 antibody.

5. The method of any one of the preceding embodiments, wherein the TypeII anti-CD20 antibody comprises a heavy chain variable region comprisingthe heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5,and the HCDR3 of SEQ ID NO: 6; and a light chain variable regioncomprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 ofSEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.

6. The method of any one of the preceding embodiments, wherein the TypeII anti-CD20 antibody comprises the heavy chain variable region sequenceof SEQ ID NO: 10 and the light chain variable region sequence of SEQ IDNO: 11.

7. The method of any one of the preceding embodiments, wherein the TypeII anti-CD20 antibody is an IgG antibody, particularly an IgG₁ antibody.

8. The method of any one of the preceding embodiments, wherein the TypeII anti-CD20 antibody is engineered to have an increased proportion ofnon-fucosylated oligosaccharides in the Fc region as compared to anon-engineered antibody.

9. The method of any one of the preceding embodiments, wherein at leastabout 40% of the N-linked oligosaccharides in the Fc region of the TypeII anti-CD20 antibody are non-fucosylated.

10. The method of any one of the preceding embodiments, wherein the TypeII anti-CD20 antibody is obinutuzumab.

11. The method of any one of the preceding embodiments, wherein thetherapeutic agent comprises a polypeptide.

12. The method of any one of the preceding embodiments, wherein thetherapeutic agent comprises an antibody.

13. The method of embodiment 12, wherein the antibody comprised in thetherapeutic agent specifically binds to carcinoembryonic antigen (CEA).

14. The method of embodiment 13, wherein the antibody comprised in thetherapeutic agent comprises a heavy chain variable region comprising theheavy chain CDR (HCDR) 1 of SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15,and the HCDR3 of SEQ ID NO: 16; and a light chain variable regioncomprising the light chain CDR (LCDR) 1 of SEQ ID NO: 17, the LCDR2 ofSEQ ID NO: 18 and the LCDR3 of SEQ ID NO: 19.

15. The method of embodiment 13 or 14, wherein the antibody comprised inthe therapeutic agent comprises the heavy chain variable region sequenceof SEQ ID NO: 20 and the light chain variable region sequence of SEQ IDNO: 21.

16. The method of embodiment 12, wherein the antibody comprised in thetherapeutic agent specifically binds to CD3, particularly CDR.

17. The method of embodiment 16, wherein the antibody comprised in thetherapeutic agent comprises a heavy chain variable region comprising theheavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33,and the HCDR3 of SEQ ID NO: 34; and a light chain variable regioncomprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 ofSEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37.

18. The method of embodiment 16 or 17, wherein the antibody comprised inthe therapeutic agent comprises the heavy chain variable region sequenceof SEQ ID NO: 38 and the light chain variable region sequence of SEQ IDNO: 39.

19. The method of any one of embodiments 1 to 18, wherein thetherapeutic agent comprises a cytokine.

20. The method of embodiment 19, wherein the cytokine is interleukin-2(IL-2).

21. The method of embodiment 19 or 20, wherein the cytokine is a mutanthuman IL-2 polypeptide comprising the amino acid substitutions F42A,Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO:12).

22. The method of any one of the preceding embodiments, wherein thetherapeutic agent comprises an immunoconjugate.

23. The method of embodiment 22, wherein the immunoconjugate comprisesan antibody as defined in any one of embodiments 13 to 15, and acytokine as defined in embodiment 20 or 21.

24. The method of any one of the preceding embodiments, wherein thetherapeutic agent comprises cergutuzumab amunaleukin (CEA-IL2v).

25. The method of any one of embodiments 1 to 18, wherein thetherapeutic agent comprises a bispecific antibody comprising an antibodyas defined in any one of embodiments 13 to 15 and an antibody as definedin any one of embodiments 16 to 18.

26. A Type II anti-CD20 antibody for use in a method of treating adisease in a subject, the method comprising a treatment regimencomprising

-   -   (i) administration to the subject of the Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (ii) administration to the subject of a therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent is sufficient for reduction of the number of        B-cells in the subject in response to the administration of the        Type II anti-CD20 antibody.

27. The Type II anti-CD20 antibody of embodiment 26, wherein thetreatment regimen effectively reduces the formation of anti-drugantibodies (ADAs) in the subject in response to the administration ofthe therapeutic agent as compared to a corresponding treatment regimenwithout the administration of the Type II anti-CD20 antibody.

28. A Type II anti-CD20 antibody for use in a method for reducing theformation of anti-drug antibodies (ADAs) against a therapeutic agent ina subject, comprising administration of the Type II anti-CD20 antibodyto the subject prior to administration of the therapeutic agent.

29. The Type II anti-CD20 antibody of embodiment 28, wherein the periodof time between the administration of the Type II anti-CD20 antibody andadministration of the therapeutic agent is sufficient for reduction ofthe number of B-cells in the subject in response to the administrationof the Type II anti-CD20 antibody.

30. The Type II anti-CD20 antibody of any one of embodiments 26 to 29,wherein the Type II anti-CD20 antibody comprises a heavy chain variableregion comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, theHCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light chainvariable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7,the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.

31. The Type II anti-CD20 antibody of any one of embodiments 26 to 30,wherein the Type II anti-CD20 antibody comprises the heavy chainvariable region sequence of SEQ ID NO: 10 and the light chain variableregion sequence of SEQ ID NO: 11.

32. The Type II anti-CD20 antibody of any one of embodiments 26 to 31,wherein the Type II anti-CD20 antibody is an IgG antibody, particularlyan IgG₁ antibody.

33. The Type II anti-CD20 antibody of any one of embodiments 26 to 32,wherein the Type II anti-CD20 antibody is engineered to have anincreased proportion of non-fucosylated oligosaccharides in the Fcregion as compared to a non-engineered antibody.

34. The Type II anti-CD20 antibody of any one of embodiments 26 to 33,wherein at least about 40% of the N-linked oligosaccharides in the Fcregion of the anti-CD20 antibody are non-fucosylated.

35. The Type II anti-CD20 antibody of any one of embodiments 26 to 34,wherein the Type II anti-CD20 antibody is obinutuzumab.

36. The Type II anti-CD20 antibody of any one of embodiments 26 to 35,wherein the therapeutic agent comprises a polypeptide.

37. The Type II anti-CD20 antibody of any one of embodiments 26 to 36,wherein the therapeutic agent comprises an antibody.

38. The Type II anti-CD20 antibody of embodiment 37, wherein theantibody comprised in the therapeutic agent specifically binds tocarcinoembryonic antigen (CEA).

39. The Type II anti-CD20 antibody of embodiment 38, wherein theantibody comprised in the therapeutic agent comprises a heavy chainvariable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO:14, the HCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO: 16; and alight chain variable region comprising the light chain CDR (LCDR) 1 ofSEQ ID NO: 17, the LCDR2 of SEQ ID NO: 18 and the LCDR3 of SEQ ID NO:19.

40. The Type II anti-CD20 antibody of embodiment 38 or 39, wherein theantibody comprised in the therapeutic agent comprises the heavy chainvariable region sequence of SEQ ID NO: 20 and the light chain variableregion sequence of SEQ ID NO: 21.

41. The Type II anti-CD20 antibody of embodiment 37, wherein theantibody comprised in the therapeutic agent specifically binds to CD3,particularly CDR.

42. The Type II anti-CD20 antibody of embodiment 41, wherein theantibody comprised in the therapeutic agent comprises a heavy chainvariable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO:32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and alight chain variable region comprising the light chain CDR (LCDR) 1 ofSEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO:37.

43. The Type II anti-CD20 antibody of embodiment 41 or 42, wherein theantibody comprised in the therapeutic agent comprises the heavy chainvariable region sequence of SEQ ID NO: 38 and the light chain variableregion sequence of SEQ ID NO: 39.

44. The Type II anti-CD20 antibody of any one of embodiments 26 to 43,wherein the therapeutic agent comprises a cytokine.

45. The Type II anti-CD20 antibody of embodiment 44, wherein thecytokine is interleukin-2 (IL-2).

46. The Type II anti-CD20 antibody of embodiment 44 or 45, wherein thecytokine is a mutant human IL-2 polypeptide comprising the amino acidsubstitutions F42A, Y45A and L72G (numbering relative to the human IL-2sequence SEQ ID NO: 12).

47. The Type II anti-CD20 antibody of any one of embodiments 26 to 46,wherein the therapeutic agent comprises an immunoconjugate.

48. The Type II anti-CD20 antibody of embodiment 47, wherein theimmunoconjugate comprises an antibody as defined in any one ofembodiments 38 to 40, and a cytokine as defined in embodiment 45 or 46.

49. The Type II anti-CD20 antibody of any one of embodiments 26 to 48,wherein the therapeutic agent comprises cergutuzumab amunaleukin(CEA-IL2v).

50. The Type II anti-CD20 antibody of any one of embodiments 1 to 43,wherein the therapeutic agent comprises a bispecific antibody comprisingan antibody as defined in any one of embodiments 38 to 40 and anantibody as defined in any one of embodiments 41 to 43.

51. Use of a Type II anti-CD20 antibody in the manufacture of amedicament for reduction of the formation of anti-drug antibodies (ADAs)against a therapeutic agent in a subject,

-   -   wherein the medicament is to be used in a treatment regimen        comprising    -   (i) administration to the subject of the Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (ii) administration to the subject of a therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent is sufficient for reduction of the number of        B-cells in the subject in response to the administration of the        Type II anti-CD20 antibody.

52. Use of a therapeutic agent in the manufacture of a medicament fortreatment of a disease in a subject, wherein the treatment comprises atreatment regimen comprising

-   -   (i) administration to the subject of a Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (ii) administration to the subject of the therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent is sufficient for reduction of the number of        B-cells in the subject in response to the administration of the        Type II anti-CD20 antibody.

53. The use of embodiment 51 or 52, wherein the treatment regimeneffectively reduces the formation of anti-drug antibodies (ADAs) againstthe therapeutic agent in the subject as compared to a correspondingtreatment regimen without the administration of the Type II anti-CD20antibody.

54. The use of any one of embodiments 51 to 53, wherein the Type IIanti-CD20 antibody comprises a heavy chain variable region comprisingthe heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5,and the HCDR3 of SEQ ID NO: 6; and a light chain variable regioncomprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 ofSEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.

55. The use of any one of embodiments 51 to 54, wherein the Type IIanti-CD20 antibody comprises the heavy chain variable region sequence ofSEQ ID NO: 10 and the light chain variable region sequence of SEQ ID NO:11.

56. The use of any one of embodiments 51 to 55, wherein the Type IIanti-CD20 antibody is an IgG antibody, particularly an IgG₁ antibody.

57. The use of any one of embodiments 51 to 56, wherein the Type IIanti-CD20 antibody is engineered to have an increased proportion ofnon-fucosylated oligosaccharides in the Fc region as compared to anon-engineered antibody.

58. The use of any one of embodiments 51 to 57, wherein at least about40% of the N-linked oligosaccharides in the Fc region of the Type IIanti-CD20 antibody are non-fucosylated.

59. The use of any one of embodiments 51 to 58, wherein the Type IIanti-CD20 antibody is obinutuzumab.

60. The use of any one of embodiments 51 to 59, wherein the therapeuticagent comprises a polypeptide.

61. The use of any one of embodiments 51 to 60, wherein the therapeuticagent comprises an antibody.

62. The use of embodiment 61, wherein the antibody comprised in thetherapeutic agent specifically binds to carcinoembryonic antigen (CEA).

63. The use of embodiment 62, wherein the antibody comprised in thetherapeutic agent comprises a heavy chain variable region comprising theheavy chain CDR (HCDR) 1 of SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15,and the HCDR3 of SEQ ID NO: 16; and a light chain variable regioncomprising the light chain CDR (LCDR) 1 of SEQ ID NO: 17, the LCDR2 ofSEQ ID NO: 18 and the LCDR3 of SEQ ID NO: 19.

64. The use of embodiment 62 or 63, wherein the antibody comprised inthe therapeutic agent comprises the heavy chain variable region sequenceof SEQ ID NO: 20 and the light chain variable region sequence of SEQ IDNO: 21.

65. The use of embodiment 61, wherein the antibody comprised in thetherapeutic agent specifically binds to CD3, particularly CDR.

66. The use of embodiment 65, wherein the antibody comprised in thetherapeutic agent comprises a heavy chain variable region comprising theheavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33,and the HCDR3 of SEQ ID NO: 34; and a light chain variable regioncomprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 ofSEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37.

67. The use of embodiment 65 or 66, wherein the antibody comprised inthe therapeutic agent comprises the heavy chain variable region sequenceof SEQ ID NO: 38 and the light chain variable region sequence of SEQ IDNO: 39.

68. The use of any one of embodiments 51 to 67, wherein the therapeuticagent comprises a cytokine.

69. The use of embodiment 68, wherein the cytokine is interleukin-2(IL-2).

70. The use of embodiment 68 or 69, wherein the cytokine is a mutanthuman IL-2 polypeptide comprising the amino acid substitutions F42A,Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO:12).

71. The use of any one of embodiments 51 to 70, wherein the therapeuticagent comprises an immunoconjugate.

72. The use of embodiment 71, wherein the immunoconjugate comprises anantibody as defined in any one of embodiments 62 to 64, and a cytokineas defined in embodiment 69 or 70.

73. The use of any one of embodiments 51 to 72, wherein the therapeuticagent comprises cergutuzumab amunaleukin (CEA-IL2v).

74. The use of any one of embodiments 51 to 67, wherein the therapeuticagent comprises a bispecific antibody comprising an antibody as definedin any one of embodiments 62 to 64 and an antibody as defined in any oneof embodiments 65 to 67.

75. A kit for the reduction of the formation of anti-drug antibodies(ADAs) against a therapeutic agent in a subject, comprising a packagecomprising a Type II anti-CD20 antibody composition and instructions forusing the Type II anti-CD20 antibody composition in a treatment regimencomprising

-   -   (iii) administration to the subject of the Type II anti-CD20        antibody composition,    -   and consecutively after a period of time    -   (iv) administration to the subject of a therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody composition and the administration of        the therapeutic agent is sufficient for reduction of the number        of B-cells in the subject in response to the administration of        the Type II anti-CD20 antibody.

76. The kit of embodiment 75, further comprising a therapeutic agentcomposition.

77. A kit for the treatment of a disease in a subject, comprising apackage comprising a therapeutic agent composition and instructions forusing the therapeutic agent composition in a treatment regimencomprising

-   -   (iii) administration to the subject of a Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (iv) administration to the subject of the therapeutic agent        composition,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent composition is sufficient for reduction of the        number of B-cells in the subject in response to the        administration of the Type II anti-CD20 antibody.

78. The kit of embodiment 77, further comprising a Type II anti-CD20antibody composition.

79. The kit of any one of embodiments 75 to 78, wherein the treatmentregimen effectively reduces the formation of anti-drug antibodies (ADAs)against the therapeutic agent in the subject as compared to acorresponding treatment regimen without the administration of the TypeII anti-CD20 antibody composition.

80. The kit of any one of embodiments 75 to 79, wherein the Type IIanti-CD20 antibody comprises a heavy chain variable region comprisingthe heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5,and the HCDR3 of SEQ ID NO: 6; and a light chain variable regioncomprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 ofSEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.

81. The kit of any one of embodiments 75 to 80, wherein the Type IIanti-CD20 antibody comprises the heavy chain variable region sequence ofSEQ ID NO: 10 and the light chain variable region sequence of SEQ ID NO:11.

82. The kit of any one of embodiments 75 to 81, wherein the Type IIanti-CD20 antibody is an IgG antibody, particularly an IgG₁ antibody.

83. The kit of any one of embodiments 75 to 82, wherein the Type IIanti-CD20 antibody is engineered to have an increased proportion ofnon-fucosylated oligosaccharides in the Fc region as compared to anon-engineered antibody.

84. The kit of any one of embodiments 75 to 83, wherein at least about40% of the N-linked oligosaccharides in the Fc region of the Type IIanti-CD20 antibody are non-fucosylated.

85. The kit of any one of embodiments 75 to 84, wherein the Type IIanti-CD20 antibody is obinutuzumab.

86. The kit of any one of embodiments 75 to 85, wherein the therapeuticagent comprises a polypeptide.

87. The kit of any one of embodiments 75 to 86, wherein the therapeuticagent comprises an antibody.

88. The kit of embodiment 87, wherein the antibody comprised in thetherapeutic agent specifically binds to carcinoembryonic antigen (CEA).

89. The kit of embodiment 88, wherein the antibody comprised in thetherapeutic agent comprises a heavy chain variable region comprising theheavy chain CDR (HCDR) 1 of SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15,and the HCDR3 of SEQ ID NO: 16; and a light chain variable regioncomprising the light chain CDR (LCDR) 1 of SEQ ID NO: 17, the LCDR2 ofSEQ ID NO: 18 and the LCDR3 of SEQ ID NO: 19.

90. The kit of embodiment 88 or 89, wherein the antibody comprised inthe therapeutic agent comprises the heavy chain variable region sequenceof SEQ ID NO: 20 and the light chain variable region sequence of SEQ IDNO: 21.

91. The kit of embodiment 87, wherein the antibody comprised in thetherapeutic agent specifically binds to CD3, particularly CDR.

92. The kit of embodiment 91, wherein the antibody comprised in thetherapeutic agent comprises a heavy chain variable region comprising theheavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33,and the HCDR3 of SEQ ID NO: 34; and a light chain variable regioncomprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 ofSEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37.

93. The kit of embodiment 91 or 92, wherein the antibody comprised inthe therapeutic agent comprises the heavy chain variable region sequenceof SEQ ID NO: 38 and the light chain variable region sequence of SEQ IDNO: 39.

94. The kit of any one of embodiments 75 to 93, wherein the therapeuticagent comprises a cytokine.

95. The kit of embodiment 94, wherein the cytokine is interleukin-2(IL-2).

96. The kit of embodiment 94 or 95, wherein the cytokine is a mutanthuman IL-2 polypeptide comprising the amino acid substitutions F42A,Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO:12).

97. The kit of any one of embodiments 75 to 96, wherein the therapeuticagent comprises an immunoconjugate.

98. The kit of embodiment 97, wherein the immunoconjugate comprises anantibody as defined in any one of embodiments 88 to 90, and a cytokineas defined in embodiment 95 or 96.

99. The kit of any one of embodiments 75 to 98, wherein the therapeuticagent comprises cergutuzumab amunaleukin (CEA-IL2v).

100. The kit of any one of embodiments 75 to 93, wherein the therapeuticagent comprises a bispecific antibody comprising an antibody as definedin any one of embodiments 88 to 90 and an antibody as defined in any oneof embodiments 91 to 93.

101. A therapeutic agent for use in a method of treating a disease in asubject, the method comprising a treatment regimen comprising

-   -   (i) administration to the subject of a Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (ii) administration to the subject of the therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent is sufficient for reduction of the number of        B-cells in the subject in response to the administration of the        Type II anti-CD20 antibody.

102. The therapeutic agent of embodiment 101, wherein the treatmentregimen effectively reduces the formation of anti-drug antibodies (ADAs)in the subject in response to the administration of the therapeuticagent as compared to a corresponding treatment regimen without theadministration of the Type II anti-CD20 antibody.

103. The therapeutic agent of embodiment 101 or 102, wherein the Type IIanti-CD20 antibody comprises a heavy chain variable region comprisingthe heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5,and the HCDR3 of SEQ ID NO: 6; and a light chain variable regioncomprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 ofSEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.

104. The therapeutic agent of any one of embodiments 101 to 103, whereinthe Type II anti-CD20 antibody comprises the heavy chain variable regionsequence of SEQ ID NO: 10 and the light chain variable region sequenceof SEQ ID NO: 11.

105. The therapeutic agent of any one of embodiments 101 to 104, whereinthe Type II anti-CD20 antibody is an IgG antibody, particularly an IgG₁antibody.

106. The therapeutic agent of any one of embodiments 101 to 105, whereinthe Type II anti-CD20 antibody is engineered to have an increasedproportion of non-fucosylated oligosaccharides in the Fc region ascompared to a non-engineered antibody.

107. The therapeutic agent of any one of embodiments 101 to 106, whereinat least about 40% of the N-linked oligosaccharides in the Fc region ofthe Type II anti-CD20 antibody are non-fucosylated.

108. The therapeutic agent of any one of embodiments 101 to 107, whereinthe Type II anti-CD20 antibody is obinutuzumab.

109. The therapeutic agent of any one of embodiments 101 to 108, whereinthe therapeutic agent comprises a polypeptide.

110. The therapeutic agent of any one of embodiments 101 to 109, whereinthe therapeutic agent comprises an antibody.

111. The therapeutic agent of embodiment 110, wherein the antibodycomprised in the therapeutic agent specifically binds tocarcinoembryonic antigen (CEA).

112. The therapeutic agent of embodiment 111, wherein the antibodycomprised in the therapeutic agent comprises a heavy chain variableregion comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 14, theHCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO: 16; and a lightchain variable region comprising the light chain CDR (LCDR) 1 of SEQ IDNO: 17, the LCDR2 of SEQ ID NO: 18 and the LCDR3 of SEQ ID NO: 19.

113. The therapeutic agent of embodiment 110 or 111, wherein theantibody comprised in the therapeutic agent comprises the heavy chainvariable region sequence of SEQ ID NO: 20 and the light chain variableregion sequence of SEQ ID NO: 21.

114. The therapeutic agent of embodiment 110, wherein the antibodycomprised in the therapeutic agent specifically binds to CD3,particularly CDR.

115. The therapeutic agent of embodiment 114, wherein the antibodycomprised in the therapeutic agent comprises a heavy chain variableregion comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, theHCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a lightchain variable region comprising the light chain CDR (LCDR) 1 of SEQ IDNO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37.

116. The therapeutic agent of embodiment 114 or 115, wherein theantibody comprised in the therapeutic agent comprises the heavy chainvariable region sequence of SEQ ID NO: 38 and the light chain variableregion sequence of SEQ ID NO: 39.

117. The therapeutic agent of any one of embodiments 101 to 116, whereinthe therapeutic agent comprises a cytokine.

118. The therapeutic agent of embodiment 117, wherein the cytokine isinterleukin-2 (IL-2).

119. The therapeutic agent of embodiment 117 or 118, wherein thecytokine is a mutant human IL-2 polypeptide comprising the amino acidsubstitutions F42A, Y45A and L72G (numbering relative to the human IL-2sequence SEQ ID NO: 12).

120. The therapeutic agent of any one of embodiments 101 to 119, whereinthe therapeutic agent comprises an immunoconjugate.

121. The therapeutic agent of embodiment 120, wherein theimmunoconjugate comprises an antibody as defined in any one ofembodiments 111 to 113, and a cytokine as defined in embodiment 118 or119.

122. The therapeutic agent of any one of embodiments 101 to 121, whereinthe therapeutic agent comprises cergutuzumab amunaleukin (CEA-IL2v).

123. The therapeutic agent of any one of embodiments 101 to 119, whereinthe therapeutic agent comprises a bispecific antibody comprising anantibody as defined in any one of embodiments 111 to 113 and an antibodyas defined in any one of embodiments 114 to 116.

In the following, further embodiments of the invention are listed.

1. A method of treating a disease in a subject, the method comprising atreatment regimen comprising

-   -   (i) administration to the subject of a Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (ii) administration to the subject of a T-cell activating        therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent is sufficient for reduction of the number of        B-cells in the subject in response to the administration of the        CD20 antibody.

2. The method of embodiment 1, wherein the treatment regimen effectivelyreduces cytokine release in the subject associated with theadministration of the therapeutic agent as compared to a correspondingtreatment regimen without the administration of the Type II anti-CD20antibody.

3. A method for reducing cytokine release associated with theadministration of a therapeutic agent in a subject, comprisingadministration of a Type II anti-CD20 antibody to the subject prior toadministration of the therapeutic agent.

4. The method of embodiment 3, wherein the period of time between theadministration of the Type II anti-CD20 antibody and administration ofthe therapeutic agent is sufficient for reduction of the number ofB-cells in the subject in response to the administration of the Type IIanti-CD20 antibody.

5. The method of any one of the preceding embodiments, wherein the TypeII anti-CD20 antibody comprises a heavy chain variable region comprisingthe heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5,and the HCDR3 of SEQ ID NO: 6; and a light chain variable regioncomprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 ofSEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.

6. The method of any one of the preceding embodiments, wherein the TypeII anti-CD20 antibody comprises the heavy chain variable region sequenceof SEQ ID NO: 10 and the light chain variable region sequence of SEQ IDNO: 11.

7. The method of any one of the preceding embodiments, wherein the TypeII anti-CD20 antibody is an IgG antibody, particularly an IgG₁ antibody.

8. The method of any one of the preceding embodiments, wherein the TypeII anti-CD20 antibody is engineered to have an increased proportion ofnon-fucosylated oligosaccharides in the Fc region as compared to anon-engineered antibody.

9. The method of any one of the preceding embodiments, wherein at leastabout 40% of the N-linked oligosaccharides in the Fc region of the TypeII anti-CD20 antibody are non-fucosylated.

10. The method of any one of the preceding embodiments, wherein the TypeII anti-CD20 antibody is obinutuzumab.

11. The method of any one of the preceding embodiments, wherein thetherapeutic agent comprises an antibody, particularly a multispecificantibody.

12. The method of embodiment 11, wherein the antibody comprised in thetherapeutic agent specifically binds to an activating T cell antigen,particularly an antigen selected from the group consisting of CD3, CD28,CD137 (also known as 4-1BB), CD40, CD226, OX40, GITR, CD27, HVEM, andCD127, more particularly CD3, most particularly CDR.

13. The method of embodiment 11 or 12, wherein the antibody comprised inthe therapeutic agent comprises a heavy chain variable region comprisingthe heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO:33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable regioncomprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 ofSEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37.

14. The method of any one of embodiments 11 to 13, wherein the antibodycomprised in the therapeutic agent comprises the heavy chain variableregion sequence of SEQ ID NO: 38 and the light chain variable regionsequence of SEQ ID NO: 39.

15. The method of any one of embodiments 11 to 14, wherein the antibodycomprised in the therapeutic agent specifically binds to a B-cellantigen, particularly an antigen selected from the group consisting ofCD20, CD19, CD22, ROR-1, CD37 and CD5, more particularly CD20 or CD19,most particularly CD20.

16. The method of embodiment 15, wherein the antibody comprised in thetherapeutic agent comprises a heavy chain variable region comprising theheavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, andthe HCDR3 of SEQ ID NO: 6; and a light chain variable region comprisingthe light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8and the LCDR3 of SEQ ID NO: 9.

17. The method of embodiment 15 or 16, wherein the antibody comprised inthe therapeutic agent comprises the heavy chain variable region sequenceof SEQ ID NO: 10 and the light chain variable region sequence of SEQ IDNO: 11.

18. The method of any one of the preceding embodiments, wherein theantibody comprised in the therapeutic agent is a bispecific antibodycomprising (i) an antibody as defined in any one of embodiments 12 to 14and (ii) an antibody as defined .in any one of embodiments 15 to 17.

19. The method of any one of the preceding embodiments, wherein thetherapeutic agent comprises CD20×CD3 bsAB.

20. The method of any one of embodiments 1 to 10, wherein thetherapeutic agent comprises a T cell expressing a chimeric antigenreceptor (CAR), particularly a CAR that specifically binds to a B-cellantigen, more particularly a CAR that specifically binds to an antigenselected from the group of CD20, CD19, CD22, ROR-1, CD37 and CD5.

21. The method of any one of the preceding embodiments, wherein thedisease is a B cell proliferative disorder, particularly a CD20-positiveB-cell disorder, and/or is a disease selected from the group consistingof Non-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL), chroniclymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL),follicular lymphoma (FL), mantle-cell lymphoma (MCL), marginal zonelymphoma (MZL), Multiple myeloma (MM) and Hodgkin lymphoma (HL).

22. A Type II anti-CD20 antibody for use in a method of treating adisease in a subject, the method comprising a treatment regimencomprising

-   -   (i) administration to the subject of the Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (ii) administration to the subject of a T-cell activating        therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent is sufficient for reduction of the number of        B-cells in the subject in response to the administration of the        Type II anti-CD20 antibody.

23. The Type II anti-CD20 antibody of embodiment 22, wherein thetreatment regimen effectively reduces cytokine release in the subjectassociated with the administration of the therapeutic agent as comparedto a corresponding treatment regimen without the administration of theType II anti-CD20 antibody.

24. A Type II anti-CD20 antibody for use in a method for reducingcytokine release associated with the administration of a therapeuticagent in a subject, comprising administration of the Type II anti-CD20antibody to the subject prior to administration of the therapeuticagent.

25. The Type II anti-CD20 antibody of embodiment 24, wherein the periodof time between the administration of the Type II anti-CD20 antibody andadministration of the therapeutic agent is sufficient for reduction ofthe number of B-cells in the subject in response to the administrationof the Type II anti-CD20 antibody.

26. The Type II anti-CD20 antibody of any one of embodiments 22 to 25,wherein the Type II anti-CD20 antibody comprises a heavy chain variableregion comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, theHCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light chainvariable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7,the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.

27. The Type II anti-CD20 antibody of any one of embodiments 22 to 26,wherein the Type II anti-CD20 antibody comprises the heavy chainvariable region sequence of SEQ ID NO: 10 and the light chain variableregion sequence of SEQ ID NO: 11.

28. The Type II anti-CD20 antibody of any one of embodiments 22 to 27,wherein the Type II anti-CD20 antibody is an IgG antibody, particularlyan IgG₁ antibody.

29. The Type II anti-CD20 antibody of any one of embodiments 22 to 28,wherein the Type II anti-CD20 antibody is engineered to have anincreased proportion of non-fucosylated oligosaccharides in the Fcregion as compared to a non-engineered antibody.

30. The Type II anti-CD20 antibody of any one of embodiments 22 to 29,wherein at least about 40% of the N-linked oligosaccharides in the Fcregion of the Type II anti-CD20 antibody are non-fucosylated.

31. The Type II anti-CD20 antibody of any one of embodiments 22 to 30,wherein the Type II anti-CD20 antibody is obinutuzumab.

32. The Type II anti-CD20 antibody of any one of embodiments 22 to 31,wherein the therapeutic agent comprises an antibody, particularly amultispecific antibody.

33. The Type II anti-CD20 antibody of embodiment 32, wherein theantibody comprised in the therapeutic agent specifically binds to anactivating T cell antigen, particularly an antigen selected from thegroup consisting of CD3, CD28, CD137 (also known as 4-1BB), CD40, CD226,OX40, GITR, CD27, HVEM, and CD127, more particularly CD3, mostparticularly CD3ε.

34. The Type II anti-CD20 antibody of embodiment 32 or 33, wherein theantibody comprised in the therapeutic agent comprises a heavy chainvariable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO:32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and alight chain variable region comprising the light chain CDR (LCDR) 1 ofSEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO:37.

35. The Type II anti-CD20 antibody of any one of embodiments 32 to 34,wherein the antibody comprised in the therapeutic agent comprises theheavy chain variable region sequence of SEQ ID NO: 38 and the lightchain variable region sequence of SEQ ID NO: 39.

36. The Type II anti-CD20 antibody of any one of embodiments 32 to 35,wherein the antibody comprised in the therapeutic agent specificallybinds to a B-cell antigen, particularly an antigen selected from thegroup consisting of CD20, CD19, CD22, ROR-1, CD37 and CD5, moreparticularly CD20 or CD19, most particularly CD20.

37. The Type II anti-CD20 antibody of embodiment 36, wherein theantibody comprised in the therapeutic agent comprises a heavy chainvariable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4,the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a lightchain variable region comprising the light chain CDR (LCDR) 1 of SEQ IDNO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.

38. The Type II anti-CD20 antibody of embodiment 36 or 37, wherein theantibody comprised in the therapeutic agent comprises the heavy chainvariable region sequence of SEQ ID NO: 10 and the light chain variableregion sequence of SEQ ID NO: 11.

39. The Type II anti-CD20 antibody of any one of embodiments 22 to 38,wherein the antibody comprised in the therapeutic agent is a bispecificantibody comprising (i) an antibody as defined in any one of embodiments33 to 35 and (ii) an antibody as defined in any one of embodiments 36 to38.

40. The Type II anti-CD20 antibody of any one of embodiments 22 to 39,wherein the therapeutic agent comprises CD20×CD3 bsAB.

41. The Type II anti-CD20 antibody of any one of embodiments 22 to 31,wherein the therapeutic agent comprises a T cell expressing a chimericantigen receptor (CAR), particularly a CAR that specifically binds to aB-cell antigen, more particularly a CAR that specifically binds to anantigen selected from the group of CD20, CD19, CD22, ROR-1, CD37 andCD5.

42. The Type II anti-CD20 antibody of any one of embodiments 22 to 41,wherein the disease is a B cell proliferative disorder, particularly aCD20-positive B-cell disorder, and/or is a disease selected from thegroup consisting of Non-Hodgkin lymphoma (NHL), acute lymphocyticleukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-celllymphoma (DLBCL), follicular lymphoma (FL), mantle-cell lymphoma (MCL),marginal zone lymphoma (MZL), Multiple myeloma (MM) and Hodgkin lymphoma(HL).

43. Use of a Type II anti-CD20 antibody in the manufacture of amedicament for reduction of cytokine release associated with theadministration of a T-cell activating therapeutic agent in a subject,wherein the medicament is to be used in a treatment regimen comprising

-   -   (i) administration to the subject of the Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (ii) administration to the subject of a T-cell activating        therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent is sufficient for reduction of the number of        B-cells in the subject in response to the administration of the        CD20 antibody.

44. Use of a T-cell activating therapeutic agent in the manufacture of amedicament for treatment of a disease in a subject, wherein thetreatment comprises a treatment regimen comprising

-   -   (iii) administration to the subject of a Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (iv) administration to the subject of the T-cell activating        therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent is sufficient for reduction of the number of        B-cells in the subject in response to the administration of the        CD20 antibody.

45. The use of embodiment 43 or 44, wherein the treatment regimeneffectively reduces cytokine release associated with the administrationof the therapeutic agent in the subject as compared to a correspondingtreatment regimen without the administration of the Type II anti-CD20antibody.

46. The use of any one of embodiments 43 to 45, wherein the Type IIanti-CD20 antibody comprises a heavy chain variable region comprisingthe heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5,and the HCDR3 of SEQ ID NO: 6; and a light chain variable regioncomprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 ofSEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.

47. The use of any one of embodiments 43 to 46, wherein the Type IIanti-CD20 antibody comprises the heavy chain variable region sequence ofSEQ ID NO: 10 and the light chain variable region sequence of SEQ ID NO:11.

48. The use of any one of embodiments 43 to 47, wherein the Type IIanti-CD20 antibody is an IgG antibody, particularly an IgG₁ antibody.

49. The use of any one of embodiments 43 to 48, wherein the Type IIanti-CD20 antibody is engineered to have an increased proportion ofnon-fucosylated oligosaccharides in the Fc region as compared to anon-engineered antibody.

50. The use of any one of embodiments 43 to 49, wherein at least about40% of the N-linked oligosaccharides in the Fc region of the Type IIanti-CD20 antibody are non-fucosylated.

51. The use of any one of embodiments 43 to 50, wherein the Type IIanti-CD20 antibody is obinutuzumab.

52. The use of any one of embodiments 43 to 51, wherein the therapeuticagent comprises an antibody, particularly a multispecific antibody.

53. The use of embodiment 51, wherein the antibody comprised in thetherapeutic agent specifically binds to an activating T cell antigen,particularly an antigen selected from the group consisting of CD3, CD28,CD137 (also known as 4-1BB), CD40, CD226, OX40, GITR, CD27, HVEM, andCD127, more particularly CD3, most particularly CDR.

54. The use of embodiment 52 or 53, wherein the antibody comprised inthe therapeutic agent comprises a heavy chain variable region comprisingthe heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO:33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable regioncomprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 ofSEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37.

55. The use of any one of embodiments 52 to 54, wherein the antibodycomprised in the therapeutic agent comprises the heavy chain variableregion sequence of SEQ ID NO: 38 and the light chain variable regionsequence of SEQ ID NO: 39.

56. The use of any one of embodiments 52 to 55, wherein the antibodycomprised in the therapeutic agent specifically binds to a B-cellantigen, particularly an antigen selected from the group consisting ofCD20, CD19, CD22, ROR-1, CD37 and CD5, more particularly CD20 or CD19,most particularly CD20.

57. The use of embodiment 56, wherein the antibody comprised in thetherapeutic agent comprises a heavy chain variable region comprising theheavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, andthe HCDR3 of SEQ ID NO: 6; and a light chain variable region comprisingthe light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8and the LCDR3 of SEQ ID NO: 9.

58. The use of embodiment 56 or 57, wherein the antibody comprised inthe therapeutic agent comprises the heavy chain variable region sequenceof SEQ ID NO: 10 and the light chain variable region sequence of SEQ IDNO: 11.

59. The use of any one of embodiments 43 to 58, wherein the antibodycomprised in the therapeutic agent is a bispecific antibody comprising(i) an antibody as defined in any one of embodiments 53 to 55 and (ii)an antibody as defined .in any one of embodiments 56 to 58.

60. The use of any one of embodiments 43 to 59, wherein the therapeuticagent comprises CD20×CD3 bsAB.

61. The use of any one of embodiments 43 to 51, wherein the therapeuticagent comprises a T cell expressing a chimeric antigen receptor (CAR),particularly a CAR that specifically binds to a B-cell antigen, moreparticularly a CAR that specifically binds to an antigen selected fromthe group of CD20, CD19, CD22, ROR-1, CD37 and CD5.

62. The use of any one of embodiments 43 to 61, wherein the disease is aB cell proliferative disorder, particularly a CD20-positive B-celldisorder, and/or is a disease selected from the group consisting ofNon-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL), chroniclymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL),follicular lymphoma (FL), mantle-cell lymphoma (MCL), marginal zonelymphoma (MZL), Multiple myeloma (MM) and Hodgkin lymphoma (HL).

63. A kit for the reduction of cytokine release associated with theadministration of a T-cell activating therapeutic agent in a subject,comprising a package comprising a Type II anti-CD20 antibody compositionand instructions for using the Type II anti-CD20 antibody composition ina treatment regimen comprising

-   -   (i) administration to the subject of the Type II anti-CD20        antibody composition,    -   and consecutively after a period of time    -   (ii) administration to the subject of a T-cell activating        therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody composition and the administration of        the therapeutic agent is sufficient for reduction of the number        of B-cells in the subject in response to the administration of        the CD20 antibody.

64. The kit of embodiment 63, further comprising a T-cell activatingtherapeutic agent composition.

65. A kit for the treatment of a disease in a subject, comprising apackage comprising a T-cell activating therapeutic agent composition andinstructions for using the therapeutic agent composition in a treatmentregimen comprising

-   -   (i) administration to the subject of a Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (ii) administration to the subject of the T-cell activating        therapeutic agent composition,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent composition is sufficient for reduction of the        number of B-cells in the subject in response to the        administration of the CD20 antibody.

66. The kit of embodiment 65, further comprising a Type II anti-CD20antibody composition.

67. The kit of any one of embodiments 63 to 66, wherein the treatmentregimen effectively reduces cytokine release associated with theadministration of the therapeutic agent in the subject as compared to acorresponding treatment regimen without the administration of the TypeII anti-CD20 antibody composition.

68. The kit of any one of embodiments 63 to 67, wherein the Type IIanti-CD20 antibody comprises a heavy chain variable region comprisingthe heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5,and the HCDR3 of SEQ ID NO: 6; and a light chain variable regioncomprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 ofSEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.

69. The kit of any one of embodiments 63 to 68, wherein the Type IIanti-CD20 antibody comprises the heavy chain variable region sequence ofSEQ ID NO: 10 and the light chain variable region sequence of SEQ ID NO:11.

70. The kit of any one of embodiments 63 to 69, wherein the Type IIanti-CD20 antibody is an IgG antibody, particularly an IgG₁ antibody.

71. The kit of any one of embodiments 63 to 70, wherein the Type IIanti-CD20 antibody is engineered to have an increased proportion ofnon-fucosylated oligosaccharides in the Fc region as compared to anon-engineered antibody.

72. The kit of any one of embodiments 63 to 71, wherein at least about40% of the N-linked oligosaccharides in the Fc region of the Type IIanti-CD20 antibody are non-fucosylated.

73. The kit of any one of embodiments 63 to 72, wherein the Type IIanti-CD20 antibody is obinutuzumab.

74. The kit of any one of embodiments 63 to 73, wherein the therapeuticagent comprises an antibody, particularly a multispecific antibody.

75. The kit of embodiment 74, wherein the antibody comprised in thetherapeutic agent specifically binds to an activating T cell antigen,particularly an antigen selected from the group consisting of CD3, CD28,CD137 (also known as 4-1BB), CD40, CD226, OX40, GITR, CD27, HVEM, andCD127, more particularly CD3, most particularly CDR.

76. The kit of embodiment 74 or 75, wherein the antibody comprised inthe therapeutic agent comprises a heavy chain variable region comprisingthe heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO:33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable regioncomprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 ofSEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37.

77. The kit of any one of embodiments 74 to 76, wherein the antibodycomprised in the therapeutic agent comprises the heavy chain variableregion sequence of SEQ ID NO: 38 and the light chain variable regionsequence of SEQ ID NO: 39.

78. The kit of any one of embodiments 74 to 77, wherein the antibodycomprised in the therapeutic agent specifically binds to a B-cellantigen, particularly an antigen selected from the group consisting ofCD20, CD19, CD22, ROR-1, CD37 and CD5, more particularly CD20 or CD19,most particularly CD20.

79. The kit of embodiment 78, wherein the antibody comprised in thetherapeutic agent comprises a heavy chain variable region comprising theheavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, andthe HCDR3 of SEQ ID NO: 6; and a light chain variable region comprisingthe light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8and the LCDR3 of SEQ ID NO: 9.

80. The kit of embodiment 78 or 79, wherein the antibody comprised inthe therapeutic agent comprises the heavy chain variable region sequenceof SEQ ID NO: 10 and the light chain variable region sequence of SEQ IDNO: 11.

81. The kit of any one of embodiments 78 to 80, wherein the antibodycomprised in the therapeutic agent is a bispecific antibody comprising(i) an antibody as defined in any one of embodiments 75 to 77 and (ii)an antibody as defined in any one of embodiments 78 to 80.

82. The kit of any one of embodiments 63 to 81, wherein the therapeuticagent comprises CD20×CD3 bsAB.

83. The kit of any one of embodiments 63 to 73, wherein the therapeuticagent comprises a T cell expressing a chimeric antigen receptor (CAR),particularly a CAR that specifically binds to a B-cell antigen, moreparticularly a CAR that specifically binds to an antigen selected fromthe group of CD20, CD19, CD22, ROR-1, CD37 and CD5.

84. The kit of any one of embodiments 63 to 83, wherein the disease is aB cell proliferative disorder, particularly a CD20-positive B-celldisorder, and/or is a disease selected from the group consisting ofNon-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL), chroniclymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL),follicular lymphoma (FL), mantle-cell lymphoma (MCL), marginal zonelymphoma (MZL), Multiple myeloma (MM) and Hodgkin lymphoma (HL).

85. A T-cell activating therapeutic agent for use in a method oftreating a disease in a subject, the method comprising a treatmentregimen comprising

-   -   (i) administration to the subject of a Type II anti-CD20        antibody,    -   and consecutively after a period of time    -   (ii) administration to the subject of the T-cell activating        therapeutic agent,    -   wherein the period of time between the administration of the        Type II anti-CD20 antibody and the administration of the        therapeutic agent is sufficient for reduction of the number of        B-cells in the subject in response to the administration of the        CD20 antibody.

86. The T-cell activating therapeutic agent of embodiment 85, whereinthe treatment regimen effectively reduces cytokine release in thesubject associated with the administration of the therapeutic agent ascompared to a corresponding treatment regimen without the administrationof the Type II anti-CD20 antibody.

87. The T-cell activating therapeutic agent of embodiment 85 or 86,wherein the Type II anti-CD20 antibody comprises a heavy chain variableregion comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, theHCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light chainvariable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7,the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.

88. The T-cell activating therapeutic agent of any one of embodiments 85to 87, wherein the Type II anti-CD20 antibody comprises the heavy chainvariable region sequence of SEQ ID NO: 10 and the light chain variableregion sequence of SEQ ID NO: 11.

89. The T-cell activating therapeutic agent of any one of embodiments 85to 88, wherein the Type II anti-CD20 antibody is an IgG antibody,particularly an IgG₁ antibody.

90. The T-cell activating therapeutic agent of any one of embodiments 85to 89, wherein the Type II anti-CD20 antibody is engineered to have anincreased proportion of non-fucosylated oligosaccharides in the Fcregion as compared to a non-engineered antibody.

91. The T-cell activating therapeutic agent of any one of embodiments 85to 90, wherein at least about 40% of the N-linked oligosaccharides inthe Fc region of the Type II anti-CD20 antibody are non-fucosylated.

92. The T-cell activating therapeutic agent of any one of embodiments 85to 91, wherein the Type II anti-CD20 antibody is obinutuzumab.

93. The T-cell activating therapeutic agent of any one of embodiments 85to 92, wherein the therapeutic agent comprises an antibody, particularlya multispecific antibody.

94. The T-cell activating therapeutic agent of embodiment 93, whereinthe antibody comprised in the therapeutic agent specifically binds to anactivating T cell antigen, particularly an antigen selected from thegroup consisting of CD3, CD28, CD137 (also known as 4-1BB), CD40, CD226,OX40, GITR, CD27, HVEM, and CD127, more particularly CD3, mostparticularly CDR.

95. The T-cell activating therapeutic agent of embodiment 93 or 94,wherein the antibody comprised in the therapeutic agent comprises aheavy chain variable region comprising the heavy chain CDR (HCDR) 1 ofSEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO:34; and a light chain variable region comprising the light chain CDR(LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 ofSEQ ID NO: 37.

96. The T-cell activating therapeutic agent of any one of embodiments 93to 95, wherein the antibody comprised in the therapeutic agent comprisesthe heavy chain variable region sequence of SEQ ID NO: 38 and the lightchain variable region sequence of SEQ ID NO: 39.

97. The T-cell activating therapeutic agent of any one of embodiments 93to 96, wherein the antibody comprised in the therapeutic agentspecifically binds to a B-cell antigen, particularly an antigen selectedfrom the group consisting of CD20, CD19, CD22, ROR-1, CD37 and CD5, moreparticularly CD20 or CD19, most particularly CD20.

98. The T-cell activating therapeutic agent of embodiment 97, whereinthe antibody comprised in the therapeutic agent comprises a heavy chainvariable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4,the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a lightchain variable region comprising the light chain CDR (LCDR) 1 of SEQ IDNO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.

99. The T-cell activating therapeutic agent of embodiment 97 or 98,wherein the antibody comprised in the therapeutic agent comprises theheavy chain variable region sequence of SEQ ID NO: 10 and the lightchain variable region sequence of SEQ ID NO: 11.

100. The T-cell activating therapeutic agent of any one of embodiments85 to 99, wherein the antibody comprised in the therapeutic agent is abispecific antibody comprising (i) an antibody as defined in any one ofembodiments 94 to 96 and (ii) an antibody as defined .in any one ofembodiments 97 to 99.

101. The T-cell activating therapeutic agent of any one of embodiments85 to 100, wherein the therapeutic agent comprises CD20×CD3 bsAB.

102. The T-cell activating therapeutic agent of any one of embodiments85 to 92, wherein the therapeutic agent comprises a T cell expressing achimeric antigen receptor (CAR), particularly a CAR that specificallybinds to a B-cell antigen, more particularly a CAR that specificallybinds to an antigen selected from the group of CD20, CD19, CD22, ROR-1,CD37 and CD5.

103. The T-cell activating therapeutic agent of any one of embodiments85 to 102, wherein the disease is a B cell proliferative disorder,particularly a CD20-positive B-cell disorder, and/or is a diseaseselected from the group consisting of Non-Hodgkin lymphoma (NHL), acutelymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuselarge B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle-celllymphoma (MCL), marginal zone lymphoma (MZL), Multiple myeloma (MM) andHodgkin lymphoma (HL).

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1 Prior Treatment with Obinutuzumab but not Rituximab or VehicleResults in the Attenuation of Tetanus Toxoid Specific De Novo IgGAntibody Responses in Cynomolgus Monkeys

To evaluate the functional impact that B-cell depletion withobinutuzumab or rituximab has on the humoral immune response to foreignantigens, cynomolgus monkeys were immune challenged after treatment witheither a novel antigen that the animals had never experienced before (denovo response to tetanus toxoid) or with a booster immune rechallengewith an immunogen that the animals had already encountered prior to theCD20 antibody administration (memory recall response tomeasles/rubella).

Animals were administered on day −14 and day −7 rituximab orobinutuzumab at a dose of 30 mg/kg or vehicle by i.v. infusion.Immunization with tetanus toxoid was performed on Day 0. Naïve animalsfrom all groups had a baseline anti-tetanus toxoid IgG measurement ofaround 0.1 IU/ml at day 0. At Day 7, vehicle treated animals andrituximab treated animals mounted robust humoral anti-tetanus toxoid IgGresponses, with an increase to around 1.0 IU/ml, while obinutuzumabtreated animals showed attenuated responses, resulting in an equal tobackground signal of 0.1 IU/ml. By Day 21, serological titers in vehicletreated and rituximab treated animals continued to rise to peak levelsof 2.5 IU/ml (FIG. 1 ). The obinutuzumab treated animals began todisplay a slight increase in titers to 0.5 IU/ml, which wassignificantly below of that of vehicle and rituximab treated groups. Theserum IgG response waned by day 51 and 68 in all groups reaching around1.5 IU/ml in vehicle, 1.3 IU/ml in rituximab and returning to 0.2 IU/mlin obinutuzumab treated animals.

These results indicate that prior treatment with obinutuzumab, but notrituximab, results in the attenuation of tetanus toxoid specific de novoIgG antibody responses in cynomolgus monkeys.

To investigate the memory recall responses by measles specific IgGantibody production in response to immune re-challenge with ameasles/rubella booster vaccination animals that had measurable baselinepositive anti-measles titers were selected. Animals were administeredrituximab or obinutuzumab at a dose of 30 mg/kg or vehicle by i.v.infusion on day −14 and Day −7. Immune re-challenge with ameasles/rubella vaccination was performed on Day 0.

Measuring the fold change at optical density OD450 nm reading overbaseline Day −14 reading, resulted in measurable increase inanti-measles titers in all three groups at Day 21, Day 51 and Day 68with no significant difference found in the IgG responses to measlesamongst the three different treatment groups (FIG. 2 ). The conclusionis that memory recall responses were left intact regardless of anti-CD20depletion therapy. The administration of either obinutuzumab orrituximab had no measurable impact on the memory recall responses to abooster immunization against measles/rubella vaccination withestablished basal titers as a result of prior vaccination.

Overall, these results showed that prior treatment with obinutuzumab ledto strong suppression of de novo antibody responses, but left theprotective humoral memory responses intact. Without wishing to be boundby theory, the ability to block de novo humoral antibody responses maypossibly be attributed to either the increased extent of endogenousB-cell depletion seen with obinutuzumab and/or the enhanced ability ofobinutuzumab to deplete activated, CD20 expressing B cells.

Example 2 A Multi-Center, Randomized, Open-Label Phase 1 Study toEvaluate Feasibility, Safety and Pharmacodynamic Effect of Pretreatmentwith Obinutuzumab Prior to Therapy with RO6895882 (CergutuzumabAmunaleukin, CEA-IL12v), in Patients with Locally Advanced and/orMetastatic Solid Tumors

Methods

An open-label, multi-center, randomized Phase 1b clinical sub-study ofRO6895882 given with or without obinutuzumab as pre-treatment isperformed.

The main objective of this sub-study is to determine if pre-treatmentwith obinutuzumab prevents the formation of ADAs in patients treatedsubsequently with RO6895882. The trial enrolls patients with locallyadvanced and/or metastatic CEA-positive solid tumors that haveprogressed on or are intolerant to the standard of care therapy.Obinutuzumab and RO6895882 are administered intravenously (IV).

Fourteen patients were randomized into the obinutuzumab pretreatment armand five patients to the control arm without obinutuzumab pretreatment.The patients in the obinutuzumab pretreatment arm received 2 g ofobinutuzumab, administered on two consecutive days (2×1000 mg), Day −13and Day −12 (+/−2 days) before the Cycle 1 Day 1 (C1D1) RO6895882administration. Pre-medication was given prior to each obinutuzumabdosing. On C1D1 all patients received a fixed and flat 10 mg dose ofRO6895882. At subsequent cycles, all patients received 20 mg ofRO6895882, administered over a minimum of 2 hour IV infusion bi-weekly(q2W). The patients in the control arm (without obinutuzumabpretreatment) received RO6895882 administrations starting at C1D1identically to the patients in the obinutuzumab pretreatment arm.

Blood samples were collected before and during the treatment period forthe monitoring of B lymphocyte counts. B cell counts were obtained usingflow cytometry and staining for CD19. Blood samples for ADAdetermination were obtained at baseline and thereafter every second weekafter the RO6895882 administration.

All patients underwent baseline and on-treatment tumor biopsies. For theobinutuzumab pretreated patients, baseline biopsies were taken afterrandomization, before administration of obinutuzumab. The controlpatients have tumor biopsies taken prior to the Cycle 1 Day 1 RO6895882administration. On-treatment tumor biopsies from the first five patientspretreated with obinutuzumab were collected on Cycle 1 Day 1 (+0/−1days) prior to RO6895882 administration in order to confirm tissue Bcell depletion before the start of RO6895882 treatment. From theremaining obinutuzumab pre-treated patients the repeated tumor biopsieswere collected on Cycle 3 Day 14 (+/−2 days), prior to the fourthRO6895882 administration on Cycle 4 Day 1. One portion of the biopsytissue was analyzed by flow cytometry and staining with CD19 for Blymphocyte detection. The second portion was formalin fixed and embeddedin paraffin and analyzed for B lymphocytes using CD20 and PAX5 staining.

The primary objectives for this study were to assess the effect ofpre-treatment with obinutuzumab on decreasing the proportion of patientswith ADA titer at cycle 4, and to evaluate the safety and tolerabilityof administration of obinutuzumab prior to treatment with RO6895882.

Secondary objectives for this study included characterization of theRO6895882 ADAs (at cycle 4) and investigation and characterization ofthe B-cell depletion in tissue (tumor and skin) and peripheral bloodresulting from obinutuzumab pre-treatment.

Results

RO6895882 (cergutuzumab amunaleukin, CEA-IL2v) has been tested in StudyBP28920 EIH Study (ClinicalTrials.gov identifier: NCT02004106). As ofNovember 2016, preliminary results from Study BP28920 show that in 59 of74 patients (80%) anti-CEA-IL2v antibodies were detected. Among the 59ADA-positive patients, 58 (98%) showed a persistent immune responsewhile 1 (1.6%) showed a transient immune response. The intensity of ADAresponses ranges from low to high titers (10-196.830), 32 out of 59persistent ADA-positive cases (54%) show high ADA titers (>100). Thefirst onset of an immune response was observed after one dose ofRO6895882 (cergutuzumab amunaleukin, CEA-IL2v) as early as on day 5,i.e. 96 h post first dose. No patient with pre-existing ADAs wasidentified and all ADAs were treatment induced. In five out of 59patients (8%), in addition to high ADA titres (>100), loss of exposurewas observed. Neither hypersensitivity reactions or signs and symptomssuggestive of hypersensitivity, nor evidence of ADA-mediated adverseevents were reported in the 108 patients who received RO6895882.

The above-described clinical sub-study was initiated to explore thepotential of obinutuzumab (Gazyva®) given as a pre-treatment toadministration of RO6895882 (cergutuzumab amunaleukin, CEA-IL2v) toattenuate development of anti-drug antibodies (ADAs). The study hasconcluded.

Fourteen out of 14 obinutuzumab treated patients remained free of ADAsagainst RO6895882 on Cycle 2. Day 1 (two weeks after first RO6895882administration) and throughout the entire study period. The longeston-treatment follow up period was up to 10 treatment cycles, i.e. 20weeks. Four out of five control patients who were not pre-treated withobinutuzumab developed ADAs with titers of 10 to 270 after 1 dose ofRO6895882.

The safety profile of obinutuzumab pre-treatment was acceptable inpatients with locally advanced and/or metastatic solid tumors. Patientsreceiving obinutuzumab/CEA-IL2v treatment did not experience unexpectedAEs compared to the CEA-IL2v monotherapy. No fatal outcome due toadverse event was reported.

Flow cytometric analysis of peripheral blood indicated completedepletion of B cells after obinutuzumab treatment. Before the start oftreatment with RO6895882 (C1D1), no B cells were detectable in thecollected blood samples (FIG. 3 ).

The tumor biopsy samples from obinutuzumab pre-treatment patients wereanalyzed by both flow cytometry and immunohistochemistry (IHC) for thefrequency of B cells. Flow cytometric analysis (FIG. 4 ) showed a strongreduction in the number and percentage of cells that stain positive forCD19, a surface protein expressed on B lymphocytes. IHC analysis of adifferent portion of the paired tumor biopsy samples detected a decreasein cells staining positive for CD20, a second antigen expressed on Bcells (FIGS. 5A and B). In order to exclude that the reduction in CD20staining was caused by obinutuzumab which may compete with anti-CD20staining, the results were confirmed by staining with PAX5, a B cellspecific transcription factor, which was also strongly reduced (FIGS. 5Cand D). Taken together, these results indicate an effective reduction ofB cells in the tumor and adjacent normal tissue.

In conclusion, the above data from this sub-study strongly suggest thatobinutuzumab is efficacious in mitigating ADAs.

Example 3 Clinical Evaluation of Feasibility, Safety and PharmacodynamicEffect of Pre-Treatment with Obinutuzumab Prior to Therapy with CEA TCB

An ongoing phase I clinical trial with CEA TCB (ClinicalTrials.govidentifier: NCT02324257) enrolls patients with locally advanced and/ormetastatic CEA-positive solid tumors who have progressed on standardtreatment, are intolerant to standard of care (SOC), and/or arenon-amenable to SOC.

Parallel cohorts of patients are opened to enroll patients that will bepretreated with obinutuzumab. The main objective of these cohorts is todetermine if pre-treatment with obinutuzumab prevents the formation ofADAs in patients treated subsequently with CEA TCB. Obinutuzumab and CEATCB are administered intravenously (IV). The patients in theobinutuzumab pre-treatment cohorts receive 2000 mg of obinutuzumab,either 2000 mg of obinutuzumab IV on Day-13 or 1000 mg of obinutuzumabIV on two consecutive days, Day-13 and Day-12 (±2 days) before the Cycle1 Day 1 (C1D1) of CEA TCB administration. Pre-medication includinganalgesic, anti-histamine and corticosteroid is given prior to eachobinutuzumab dosing. On C1D1, all patients receive a flat dose(depending on the dose cohort they are allocated to, as defined in theongoing clinical trial) of CEA TCB over a minimum of 120-minute IVinfusion. At subsequent cycles, all patients receive the same dose ofCEA TCB, administered over a minimum of 90-minute IV infusion at C2D1and over a minimum of 60-minute IV infusion from C3D1 onwards weekly(QW). The patients not receiving obinutuzumab pre-treatment receive CEATCB administrations starting at C1D1 identically to the patients in theobinutuzumab pre-treatment cohorts. Blood samples are collected beforeand during the treatment period for the monitoring of B lymphocytecounts. B cell counts are obtained using flow cytometry and staining forCD19. Blood samples for PK to evaluate the serum levels of CEA TCB andfor ADA determination are obtained at baseline and thereafter at eachcycle of CEA TCB administration. The primary objectives for thesecohorts of patients receiving obinutuzumab pre-treatment is the effectof obinutuzumab pretreatment in decreasing the rate of patients withpositive Anti-Drug Antibodies (ADA) titer against CEA TCB at week 8and/or delaying the time of onset of ADA against CEA TCB, and toevaluate the safety and tolerability of administration of obinutuzumabprior to treatment with CEA TCB. The study will also be looking at thecharacterization of the ADA directed against CEA TCB; it includes aswell investigation and characterization of the B-cell depletion intissue (tumor biopsies are undertaken at Baseline and at week 7 afterC1D1 with CEA TCB) and peripheral blood resulting from obinutuzumabpre-treatment.

Preliminary Results

CEA TCB is being tested in a EIH Study (ClinicalTrials.gov identifier:NCT02324257). As of 27 Oct. 2016 (ADA data cutoff), preliminary resultsfrom Study BP29541 show that in 40 of 77 patients (52%) anti-CEA TCBantibodies were detected, none of these patients received obinutuzumabpre-treatment, except for two patients with transient positive titers(one had a titer of 30 at Cycle 2 Day 1 but became then negative up toCycle 24 and another one had a titer of 270 at Cycle 3 Day 1 and 90 atCycle 4 Day 1 but became then negative up to Cycle 8). Except for thesetwo patients, all the 38 other ADA-positive patients showed a persistentimmune response. The intensity of ADA responses ranged from low to hightiters (10-21870). 17 out of 38 persistent ADA-positive cases (45%)showed moderate ADA titers (≤810), while 21 out of 28 persistentADA-positive cases (55%) showed high ADA titers (>810). The first onsetof an immune response was observed after one dose of CEA TCB at day 8(C2D1 pre-dose), however 3 patients had positive titers at Cycle 1 Day 1pre-dose. Three patients with pre-existing ADAs were identified and inall other patients ADAs were treatment-induced. In 24 out of 38 patients(63%) with a persistent immune response, the sustained ADA presencecorrelated with total loss of exposure (ranges from Cycle 3 Day 1 andCycle 13 Day 1 (PK data cutoff: 7 Nov. 2016)). So far nohypersensitivity reactions or signs and symptoms suggestive ofhypersensitivity, or evidence of ADA-mediated adverse events have beenreported in the 77 patients who received CEA TCB. The above-describedclinical study with parallel cohorts of patients receiving obinutuzumabpre-treatment was initiated to explore the potential of obinutuzumab(Gazyva®/Gazyvaro®) given as a pre-treatment to administration of CEATCB to attenuate development of anti-drug antibodies (ADAs). The studyis ongoing. According to the preliminary data, as of the clinical cutoffdate of 27 Oct. 2016 (ADA data cutoff), the 12 obinutuzumab pre-treatedpatients for which ADA data is available so far (2 of the patients onlyreceived obinutuzumab as they clinically deteriorated in the meantimeand did not receive any CEA TCB infusion) remained free of ADAs againstCEA TCB at the following time points: 1 patient up to C1, 1 patient upto C3, 1 patient up to C4, 1 patient up to C6, 1 patient up to C8, 1patient up to C10, 2 patients up to C12, 1 patient up to C16, and 1patient up to C25.

Eight out of sixty-five patients who were not pre-treated withobinutuzumab developed ADAs with titers of 10 to 810 after 1 dose of CEATCB. Based on a preliminary safety analysis, the safety profile ofobinutuzumab pre-treatment was acceptable in patients with locallyadvanced and/or metastatic solid tumors. Patients receiving obinutuzumabpre-treatment prior to receiving weekly doses of CEA TCB did notexperience unexpected adverse events (AEs) compared to the ones who didnot receive obinutuzumab pre-treatment. Flow cytometric analysis ofperipheral blood indicated complete depletion of B cells afterobinutuzumab treatment. Before the start of treatment with CEA TCB(C1D1), no B cells were detectable in the collected blood samples. Thetumor biopsy samples from obinutuzumab pre-treatment patients wereundertaken at Baseline prior to receiving obinutuzumab pre-treatment andon treatment at Cycle 7 Day 1. The analyses are still ongoing.

Example 4 Assessment of the Anti-Tumour Activity and Cytokine ReleaseMediated by CD20×CD3 bsAB±Obinutuzumab Pre-Treatment (Gpt) in FullyHumanized Mice

We investigated whether Gpt could prevent the cytokine releaseassociated with the first administration of CD20×CD3 bsAB in fullyhumanized NOG mice.

All treatment options (obinutuzumab, CD20×CD3 bsAB and Gpt+CD20×CD3bsAB) led to efficient peripheral blood B-cell depletion detectedalready 24 hours after the first therapy administration (FIG. 7A). Tcell counts revealed a transient decrease in the peripheral blood 24hours after the first administration of CD20×CD3 bsAB but not followingobinutuzumab or Gpt+CD20×CD3 bsAB (FIG. 7B). Therefore, whenadministered prior to CD20×CD3 bsAB, a single administration ofobinutuzumab abrogates CD20×CD3 bsAB-mediated T cell decrease in theperipheral blood.

The analysis of cytokines released in blood of treated mice in thedifferent experimental groups revealed that CD20×CD3 bsAB treatmentinduces a transient elevation of several cytokines in the blood, with apeak at 24 hours after the first administration and a return to nearbaseline levels by 72 hours (FIG. 8 ). MIP-1b, IL-5, IL-10, MCP-1 show asimilar trend to IFNγ, TNFα and IL-6 (not shown). Gpt strongly reducedthe cytokine release in the peripheral blood associated with the firstCD20×CD3 bsAB injection (Table 2).

TABLE 2 Cytokines Released in the Peripheral Blood of Fully HumanizedNOG Mice uponCD20XCD3 bsAB and Gpt + CD20XCD3 bsAB Treatments TreatmentGpt + Vehicle CD20XCD3 bsAB CD20XCD3 Cytokine (pg/ml) (pg/ml) bsAB(pg/ml) IFN-g  18.50 (18.07)   756.95 (357.30)   183.134 (171.91) TNF-a 12.47 (2.95)    79.56 (28.98)    14.89 (2.56) IL-6  15.39 (7.15)  613.27 (140.60)   178.34 (117.85) IL-8  11.44 (2.64)   292.68 (132.36)  150.58 (96.76) MIP-1b 272.70 (97.05)   2129.44 (132.36)   338.95(71.25) MCP-1  73.49 (13.89)   2146.31 (672.69)   393.29 (188.86) IL-10223.48 (62.48) 15,278.89 (6584.50)   945.04 (604.89) IL-4  0.75 (0.14)   1.99 (0.77)    0.81 (0.02) G-CSF  14.60 (5.14)    21.23 (16.36)   3.82 (2.02) GM-CSF 945.97 (155.74)   1207.48 (299.83)   626.18(282.46) IL-5  10.42 (3.35)   162.33 (140.82)    13.58 (8.44) IL-2  19.1(8.42)   369.70 (360.64)    19.59 (17.64) IL-13  5.39 (3.66)    15.42(11.18)    2.96 (1.11) IL-1b  1.48 (0.2)    6.40 (1.94)    3.47 (1.88)IL-7  6.98 (0)    4.27 (2.55)    6.17 (1.79) IL-12p40  43.59 (19.45)   51.31 (23.12)    17.05 (2.62) IL-17 194.40 (96.32)   274.79 (112.20)   73.33 (32.43) Notes: Data are displayed as the arithmetic mean (SD).N = 5 in both treatments.

The anti-tumour activity of CD20×CD3 bsAB was not affected bypre-treatment with obinutuzumab (FIG. 9 ). Obinutuzumab treatment, asmonotherapy, showed a strong anti-tumour activity, although with slowerkinetics when compared to CD20×CD3 bsAB in this tumour and mouse model.

The data therefore indicate that Gpt reduces the cytokine releaseassociated with the first CD20×CD3 bsAB injection, however, despitetargeting the same antigen on tumour cells, the anti-tumour activity ofCD20×CD3 bsAB is not affected by Gpt.

Example 5 Obinutuzumab Pre-Treatment Study in Cynomolgus Monkeys

A mechanistic study (non-GLP) in male cynomolgus monkeys was performedto investigate the effects of pretreatment with obinutuzumab on responseto CD20×CD3 bsAB at doses of 0.1, 0.3 and 1 mg/kg) (Table 3). In thisstudy, 6 naïve cynomolgus male monkeys/group (4 for Group 1), receivedan IV dose of either control article 1 (Groups 1 and 2) or obinutuzumab(50 mg/kg, Groups 3, 4, 5), followed 4 days later by treatment withcontrol article 2 (Group 1), CD20×CD3 bsAB, 0.1 mg/kg (Group 2, Group3), CD20×CD3 bsAB, 0.3 mg/kg (Group 4) or CD20×CD3 bsAB, 1 mg/kg (Group5). Four days between the obinutuzumab and CD20×CD3 bsAB dosing wasconsidered sufficient to allow depletion of B cells in peripheral blood,lymph nodes and spleen by obinutuzumab. On Day 12, 2 animals from Group1, and 4 from Groups 2 to 5 were necropsied (terminal necropsy). Twoanimals from each group were retained for an 8-week recovery period.

TABLE 3 Study Design: Obinutuzumab Pre- Treatment in Cynomolgus Monkeys.Dose Group Dosing Level Number of Males No. Test Article Day (mg/kg)Main ^(a) Recovery ^(b) 1 Control Article 1 1 0 2 2 Control Article 2 50 2 Control Article 1 1 0 4 2 CD20 × CD3 bsAB 5 0.1 3 obinutuzumab 1 504 2 CD20 × CD3 bsAB 5 0.1 4 obinutuzumab 1 50 4 2 CD20 × CD3 bsAB 5 0.35 obinutuzumab 1 50 4 2 CD20 × CD3 bsAB 5 1 Note: Control Article 1 =Control for obinutuzumab: Control article 2 = Control for CD20 × CD3bsAB. ^(a) Main group animals, terminal necropsy Day 12. ^(b) Recoveryanimals, necropsy week 8 (Day 61).

The following data are available from this study:

-   -   Following pretreatment with obinutuzumab (50 mg/kg, Gpt), IV        administration of CD20×CD3 bsAB was tolerated up to 1 mg/kg, the        highest tested dose. Clinical signs, observed with CD20×CD3 bsAB        alone (emesis, hunched posture and hypoactivity) were markedly        reduced by Gpt at all doses of CD20×CD3 bsAB.    -   CD20×CD3 bsAB administration alone resulted in the reduction of        B lymphocytes and the activation and expansion of T-lymphocyte        (CD4+ and CD8+) subsets and NK cells. In contrast, the        administration of obinutuzumab prior to CD20×CD3 bsAB        administration resulted in B-lymphocyte depletion, as well as        the subsequent attenuation of T-lymphocyte activation as        demonstrated by reductions in the transient reductions of        lymphocyte and monocyte populations after CD20×CD3 bsAB        administration, as well as reductions in T-cell activation        marker up-regulation and expansion, relative to changes present        for animals that were treated with CD20×CD3 bsAB alone.    -   The release of IFNγ, IL-8, TNFα, IL-2 and IL-6, 4-hour post-0.1        mg/kg CD20×CD3 bsAB treatment, was markedly reduced in the Gpt        groups. Similarly, low levels of cytokine release were noted at        higher doses of CD20×CD3 bsAB in Gpt groups (FIG. 10 ).    -   CD20×CD3 bsAB-related histopathologic findings were restricted        to the lymphoid organs (e.g. decreased cellularity specifically        affecting the CD20-positive cells was present in the lymphoid        follicles of the spleen). The CD20-positive cell decreases were        almost completely reversed after the 8 week treatment-free        period. No other histopathological changes were present,        including in brain, spinal cord and sciatic nerve in monkeys        treated with CD20×CD3 bsAB at 0.1 mg/kg and in animals        administered CD20×CD3 bsAB at 0.1, 0.3 or 1 mg/kg following Gpt.

Example 6 Clinical Evaluation of Safety, Tolerability andPharmacokinetics of CD20×CD3 bsAB with Obinutuzumab Pre-Treatment inPatients with r/r NHL

A phase I dose-escalation study will be performed, the primaryobjectives of which include evaluation of the safety, tolerability andpharmacokinetics CD20×CD3 bsAB with obinutuzumab pre-treatment inpatients with relapsed/refractory (r/r) NHL.

The study will enroll patients with r/r NHL, whose tumours are expectedto express CD20 in B cells. Patients with CLL will not be enrolled.Patients are expected to have relapsed after or failed to respond to atleast one prior treatment regimen.

Obinutuzumab and CD20×CD3 bsAB will be administered intravenously (IV).

Prior to administration of obinutuzumab and CD20×CD3 bsAB, premedicationwith corticosteroids (e.g., 20 mg IV dexamethasone, 80 mg IVmethylprednisone, or equivalent) will be administered, along withanti-histamines and acetaminophen. Prophylactic measures for otherevents, such as tumor lysis syndrome will also be either recommended asneeded or mandated.

CD20×CD3 bsAB will be initiated on Cycle 1/Day 1 (C1/D1) as a singleagent by intravenous (IV) infusion, following pre-treatment with asingle dose of obinutuzumab (1000 mg; IV) seven days in advance (Cycle1/Day −7) of the first CD20×CD3 bsAB dose (Cycle 1/Day 1). Theanticipated starting dose of CD20×CD3 bsAB will be 5 micrograms (flatdosing). All dosing cycles are 14 days (Q2W) long, with one additionaldose given in Cycle 1 only. Thus, the dosing scheme is foradministration of CD20×CD3 bsAB on Days 1 and 8 in Cycle 1 (C1/D1;C1/D8), followed by dosing in all subsequent Cycles on Day 1 only (Q2W)for a total of 12 cycles (24 weeks) of treatment or until unacceptabletoxicity or progression occurs.

Blood samples will be collected at appropriate timepoints to determinethe relevant PK properties of CD20×CD3 bsAB, as well as a range of PDmarkers in blood, to assess e.g. magnitude and kinetics of B-celldepletion following Gpt and CD20×CD3 bsAB dose initiation, T-cellphenotypes, and to assess soluble mediator release (cytokines andchemokines), following administration of Gpt and CD20×CD3 bsAB atselected timepoints.

Example 7 Comparison of the Anti-Tumor Activity of Step-Up Dosing (SUD)and Obinutuzumab Pretreatment (Gpt)

As shown in this example, Gpt is a superior approach to step-up dosing(SUD) in terms of anti-tumor activity and T-cell redistribution inperipheral tissues.

Fully humanized NOG female mice (Taconic) were generated in house. Agewas 20 weeks at start of experiment. All mice were injected s.c. onstudy day 0 with 1.5×10⁶ of WSU-DLCL2 cells (human diffuse large B celllymphoma). Seven days after tumor cell administration (Day 7), mice wereadministered i.v. with the first treatment as indicated in Table 4. Thesecond treatment as indicated in Table 4 was administered on Day 14.

Following 9 weeks of CD20×CD3 bsAB treatment, the number of tumor-freemice at study termination (Day 69) was higher when treatment waspreceded by obinutuzumab (Gpt) than when preceded by any of three singlefractionated doses of CD20×CD3 bsAB (SUD) (FIG. 11 , Table 4).

TABLE 4 Anti-tumor activity upon step-up dosing of CD20 × CD3 BSAB andobinutuzumab pretreatment (Gpt) in fully humanized NOG mice bearingWSU-DLCL2 tumors. Tumor-Free Mice at Termination 1^(st) Treatment (Dose)2^(nd) Treatment (Dose) (Day 69) CD20 × CD3 bsAB (0.15 CD20 × CD3 bsAB(0.5 4/9 mg/kg) mg/kg) CD20 × CD3 bsAB (0.05 CD20 × CD3 bsAB (0.5 2/9mg/kg) mg/kg) CD20 × CD3 bsAB (0.015 CD20 × CD3 bsAB (0.5 3/10 mg/kg)mg/kg) Obinutuzumab (10 mg/kg) CD20 × CD3 bsAB (0.5 7/10 mg/kg)

In addition to superior anti-tumor efficacy, seven days following thefirst (pre-)treatment (corresponding to study Day 14), an increase inperivascular CD3 positive T cells was observed in the lungs of CD20×CD3bsAB-treated mice (full dose or fraction of the full dose) but not withobinutuzumab (10 mg/kg)-treated mice (FIG. 12A-D). The same was true forthe analysis at a later time point corresponding to 24 hours after thesecond treatment (study Day 15) (FIG. 12E-H).

For this experiment, mice were generated and injected with tumor cellsas described above. Seven days after tumor cell administration, micewere administered i.v. with the treatments as indicated in Table 5A.

Four animals/group were sacrificed 7 days after receiving a single doseof CD20×CD3 bsAB, obinutuzumab or vehicle. The remaining fouranimals/group were sacrificed 24 hours after receiving the secondtreatment on Day 14, as indicated in Table 5B. Controls received thevehicle buffer.

Lung, liver and kidney were collected at necropsy and serial sectionswere stained with immunohistochemistry (IHC) for human CD3 according toestablished protocols. Sections stained immunohistochemically for CD3were evaluated blinded by a Board Certified Veterinary Pathologist usinga score from 0 (no or rare CD3 positive cells in the section) to 3 (manyCD3 positive cells surrounding the vessels/ducts).

Representative results from lung sections are shown in FIG. 12 , Table5.

A dose dependent increase in perivascular CD3 positive T cells wasobserved in the lung of mice seven days after single dose of >0.05 mg/kgCD20×CD3 bsAB (full dose or fraction of the full dose), but not inobinutuzumab-treated mice (FIG. 12A-D, Table 5A).

TABLE 5A Lung IHC for CD3 - animals sacrificed 7 days after firsttreatment. Table shows the average score of perivascular CD3 positive Tcells in the lung (performed by blinded pathologist analysis). Treatment(dose) Average Score Vehicle (−) 0.75 CD20 × CD3 bsAB 3 (0.5 mg/kg) CD20× CD3 bsAB 1.75 (0.15 mg/kg) CD20 × CD3 bsAB 1.5 (0.05 mg/kg) CD20 × CD3bsAB 0.5 (0.015 mg/kg) Obinutuzumab (10 mg/kg) 0.25

An increase in perivascular T cells was also observed in mice 24 hoursafter a single or a repeated dose of CD20×CD3 bsAB, but not in micepretreated with obinutuzumab (FIG. 12E-H, Table 5B).

TABLE 5B Lung IHC for CD3 - animals sacrificed 24 hours after 2ndtreatment. Table shows the average score of perivascular CD3 positive Tcells in the lung (performed by blinded pathologist analysis). FirstTreatment Second Treatment (dose) (dose) Average Score Vehicle (−)Vehicle (−) 0 Vehicle (−) CD20 × CD3 bsAB 1.5 (0.5 mg/kg) CD20 × CD3bsAB CD20 × CD3 bsAB 1.7 (0.15 mg/kg) (0.5 mg/kg) CD20 × CD3 bsAB CD20 ×CD3 bsAB 2.5 (0.05 mg/kg) (0.5 mg/kg) CD20 × CD3 bsAB CD20 × CD3 bsAB2.3 (0.015 mg/kg) (0.5 mg/kg) Obinutuzumab (10 CD20 × CD3 bsAB 0.5mg/kg) (0.5 mg/kg)

In animals receiving a single dose of 0.5 mg/kg of CD20×CD3 bsABsacrificed 24 hours after treatment, margination and adhesion of CD3positive T cells was present in lung vessels (FIG. 13 ), suggesting anearly activation of T cells with transmigration into the perivascularspace.

In conclusion, single or repeated treatment with full dose or fractionof the full dose of CD20×CD3 bsAB resulted in an increase in CD3positive T cells compared to controls with perivascular distribution inthe lung, perivascular/periductal in the liver and in the glomeruli inthe kidneys (liver and kidney data not shown). These changes were notoccurring in animals pretreated with obinutuzumab.

Example 8 Comparison of CD20×CD3 bsAB Exposure in Cynomolgus Monkeyswith or without Gpt

In this example, Gpt is shown to increase CD20×CD3 bsAB exposure.Cynomolgus monkeys were administered a single IV dose of 100-1000 μg/kgCD20×CD3 bsAB with or without obinutuzumab pretreatment (Gpt) (50 mg/kg,4 days prior to CD20×CD3 bsAB administration).

The increased exposure resulting from the depletion of CD20⁺ B cells byobinutuzumab (50 mg/kg) is shown by the CD20×CD3 bsAB serum levelsfollowing a single dose of 100 μg/kg CD20×CD3 bsAB with or without Gpt(FIG. 14 ). Clearance of CD20×CD3 bsAB following Gpt was markedlyreduced to about one fourth of the clearance observed in the groupwithout Gpt (Table 6). By the time of CD20×CD3 bsAB dosing, B cells werereduced by Gpt to about 1-5% of baseline levels. The reduction inclearance is consistent with the Gpt-induced B-cell depletion and theresulting reduction of target-mediated clearance from B-cell binding.Following Gpt, clearance and the central volume of distribution (Vc)were independent of dose in the dose range studied, with clearancevalues around 90 mL/day/kg and Vc values around 40 mL/kg, which issimilar to the serum volume.

TABLE 6 Mean Pharmacokinetic parameters of CD20XCD3 bsAB following asingle intravenous administration with or without obinutuzumabpretreatment in cynomolgus monkeys. Dose Obinutuzumab CL C_(max)AUC_(inf)/Dose (μg/kg) pretreatment (mL/day/kg) V_(c) (mL/kg) T_(1/2)ª(h) (μg/mL) (μg•h/mL/μg/kg)  100 No 384 43.2 7.53 2.22 0.0679 (27.7)(16.8) (50.3) (17.3) (36.3)  100 Yes 81.7 41.1 67.7 2.56 0.308 (22.4)(13.6) (34.0) (12.5) (25.1)  300 Yes 110 37.9 53.4 7.63 0.234 (28.8)(5.2) (10.4) (7.8) (29.4) 1000 Yes 87.5 34.9 54.6 28.7 0.283 (19.0)(10.0) (17.6) (9.8) (19.7) AUC_(inf) = area under the concentration-timecurve from Time 0 to infinity; CL = clearance: C_(max) = maximumconcentration; T_(1/2) = half-life; V_(c) = central volume ofdistribution. Notes: Cynomolgus monkeys were administered a single IVdose of 100-1000 μg/kg CD20XCD3 bsAB with or without obinutuzumabpretreatment (Gpt) (50 mg/kg, 5 days prior to CD20XCD3 bsABadministration) in a Mechanistic Safety Study. Values are averages ofsix animals per group ( + %CV). ^(a)Apparent T_(1/2) (due to non-linearpharmacokinetics)

Example 9 Obinutuzumab Pre-Treatment to Avoid Cytokine Release afterAdoptive T Cell Therapy with CAR-T Cells

Cytokine release syndrome (CRS) is a very frequent phenomenon followingtreatment with CD19 CAR-T cells as well as CAR-T cells directed againstCD20 or CD22 that can result in lethal side effects. Strategies to avoidor reduce CRS focus on various aspects of CAR-T therapy (reviewed in Xuand Tang, Cancer Letters (2014) 343, 172-178).

We suggest a novel approach to avoid CRS following treatment with CAR-Tcells in B cell proliferative disorders, by depletion of peripheral andmalignant B cells using obinutuzumab pre-treatment.

For this purpose, patients with a B-cell proliferative disorder (e.g.NHL) are randomized into an obinutuzumab pre-treatment arm and a controlarm without obinutuzumab pre-treatment. The patients in the obinutuzumabpre-treatment arm receive 1 g of obinutuzumab, administered on Day −7(+/−2 days) before administration of CD19, CD20 or CD22 CAR-T cells.

Patients are infused with autologous T cells transduced with a CARlentiviral vector at an appropriate dose for the specific CAR-T cellused, the patient and the disease to be treated (e.g. 0.76×10⁶ to20.6×10⁶ CAR-T cells per kilogram of body weight as described in Maudeet al., N Engl J Med (2014) 371, 1507-1517; 1.4×10⁶ to 1.2×10⁷ CAR-Tcells per kilogram of body weight as described in Grupp et al., New EnglJ Med (2013) 368, 1509-1518; or 0.14×10⁸ to 11×10⁸ CAR-T cells asdescribed in Porter et al., Sci Transl Med (2015) 7, 303ra139). Patientsare monitored for a response, toxic effects, and the expansion andpersistence of circulating CAR-T cells.

Pre-medication is given prior to each obinutuzumab dosing. Blood samplesare collected before and during the treatment period for the monitoringof B lymphocyte counts. B cell counts are obtained using flow cytometryand staining for CD19. In addition, incidence of CRS is screened bymeasuring cytokines including IL-6.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1. A Type II anti-CD20 antibody for use in a method of treating adisease in a subject, the method comprising a treatment regimencomprising (i) administration to the subject of the Type II anti-CD20antibody, and consecutively after a period of time (ii) administrationto the subject of a therapeutic agent, wherein the period of timebetween the administration of the Type II anti-CD20 antibody and theadministration of the therapeutic agent is sufficient for reduction ofthe number of B-cells in the subject in response to the administrationof the Type II anti-CD20 antibody.
 2. The Type II anti-CD20 antibody ofclaim 1, wherein the treatment regimen effectively reduces the formationof anti-drug antibodies (ADAs) in the subject in response to theadministration of the therapeutic agent as compared to a correspondingtreatment regimen without the administration of the anti-CD20 antibody.3. The Type II anti-CD20 antibody of claim 1, wherein the treatmentregimen effectively reduces cytokine release associated with theadministration of the therapeutic agent in the subject as compared to acorresponding treatment regimen without the administration of the TypeII anti-CD20 antibody.
 4. The Type II anti-CD20 antibody of claim 3,wherein the therapeutic agent is a T cell activating therapeutic agent.5. A Type II anti-CD20 antibody for use in a method for (i) reducing theformation of anti-drug antibodies (ADAs) against a therapeutic agent ina subject and/or (ii) reducing cytokine release associated with theadministration a therapeutic agent, particularly a T-cell activatingtherapeutic agent, in a subject, comprising administration of the TypeII anti-CD20 antibody to the subject prior to administration of thetherapeutic agent.
 6. The Type II anti-CD20 antibody of claim 5, whereinthe period of time between the administration of the Type II anti-CD20antibody and administration of the therapeutic agent is sufficient forreduction of the number of B-cells in the subject in response to theadministration of the Type II anti-CD20 antibody.
 7. The Type IIanti-CD20 antibody of claim 1, wherein the anti-CD20 antibody comprisesa heavy chain variable region comprising the heavy chain CDR (HCDR) 1 ofSEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6;and a light chain variable region comprising the light chain CDR (LCDR)1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO:9.
 8. The Type II anti-CD20 antibody of claim 1, wherein the anti-CD20antibody comprises the heavy chain variable region sequence of SEQ IDNO: 10 and the light chain variable region sequence of SEQ ID NO:
 11. 9.The Type II anti-CD20 antibody of claim 1, wherein the anti-CD20antibody is an IgG antibody, particularly an IgG1 antibody, and whereinat least about 40% of the N-linked oligosaccharides in the Fc region ofthe anti-CD20 antibody are non-fucosylated.
 10. The Type II anti-CD20antibody of claim 1, wherein the anti-CD20 antibody is obinutuzumab. 11.The Type II anti-CD20 antibody of claim 1, wherein the therapeutic agentcomprises a polypeptide.
 12. The Type II anti-CD20 antibody of claim 1,wherein the therapeutic agent comprises an antibody.
 13. The Type IIanti-CD20 antibody of claim 12, wherein the antibody comprised in thetherapeutic agent specifically binds to carcinoembryonic antigen (CEA).14. The Type II anti-CD20 antibody of claim 13, wherein the antibodycomprised in the therapeutic agent comprises (i) a heavy chain variableregion comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 14, theHCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO: 16; and a lightchain variable region comprising the light chain CDR (LCDR) 1 of SEQ IDNO: 17, the LCDR2 of SEQ ID NO: 18 and the LCDR3 of SEQ ID NO: 19; or(ii) a heavy chain variable region sequence of SEQ ID NO: 20 and a lightchain variable region sequence of SEQ ID NO:
 21. 15. The Type IIanti-CD20 antibody of claim 12, wherein the antibody comprised in thetherapeutic agent specifically binds to CD3, particularly CD3ε.
 16. TheType II anti-CD20 antibody of claim 15, wherein the antibody comprisedin the therapeutic agent comprises (i) a heavy chain variable regioncomprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 ofSEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chainvariable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO:35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37; or (ii) aheavy chain variable region sequence of SEQ ID NO: 38 and a light chainvariable region sequence of SEQ ID NO:
 39. 17. The Type II anti-CD20antibody of claim 12, wherein the antibody comprised in the therapeuticagent specifically binds to CD20.
 18. The Type II anti-CD20 antibody ofclaim 15, wherein the antibody comprised in the therapeutic agentcomprises (i) a heavy chain variable region comprising the heavy chainCDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3of SEQ ID NO: 6; and a light chain variable region comprising the lightchain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and theLCDR3 of SEQ ID NO: 9; or (ii) a heavy chain variable region sequence ofSEQ ID NO: 10 and a light chain variable region sequence of SEQ ID NO:11.
 19. The Type II anti-CD20 antibody of claim 1, wherein thetherapeutic agent comprises a cytokine, particularly interleukin-2. 20.The Type II anti-CD20 antibody of claim 19, wherein the cytokine is amutant human IL-2 polypeptide comprising the amino acid substitutionsF42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQID NO: 12). 21-23. (canceled)